Reducing Systemic Regulatory T Cell Levels or Activity for Treatment of Disease and Injury of the CNS

ABSTRACT

A pharmaceutical composition comprising an active agent that causes reduction of the level of systemic immunosuppression in an individual for use in treating a disease, disorder, condition or injury of the CNS that does not include the autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RRMS), is provided. The pharmaceutical composition is for administration by a dosage regimen comprising at least two courses of therapy, each course of therapy comprising in sequence a treatment session followed by an interval session.

FIELD OF THE INVENTION

The present invention relates in general to methods and compositions fortreating disease, disorder, condition or injury of the Central NervousSystem (CNS) by transiently reducing the level of systemicimmunosuppression in the circulation.

BACKGROUND OF THE INVENTION

Most central nervous system (CNS) pathologies share a commonneuroinflammatory component, which is part of disease progression, andcontributes to disease escalation. Among these pathologies isAlzheimer's disease (AD), an age-related neurodegenerative diseasecharacterized by progressive loss of memory and cognitive functions, inwhich accumulation of amyloid-beta (Aβ) peptide aggregates was suggestedto play a key role in the inflammatory cascade within the CNS,eventually leading to neuronal damage and tissue destruction (Akiyama etal, 2000; Hardy & Selkoe, 2002; Vom Berg et al, 2012). Despite thechronic neuroinflammatory response in neurodegenerative diseases,clinical and pre-clinical studies over the past decade, investigatingimmunosuppression-based therapies in neurodegenerative diseases, haveraised the question as to why anti-inflammatory drugs fall short(Breitner et al, 2009; Group et al, 2007; Wyss-Coray & Rogers, 2012). Weprovide a novel answer that overcomes the drawbacks of existingtherapies of AD and similar diseases and injuries of the CNS; thismethod is based on our unique understanding of the role of the differentcomponents of systemic and central immune system in CNS maintenance andrepair.

SUMMARY OF INVENTION

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an active agent that causes reduction of thelevel of systemic immunosuppression in an individual for use in treatinga disease, disorder, condition or injury of the CNS that does notinclude the autoimmune neuroinflammatory disease, relapsing-remittingmultiple sclerosis (RRMS), wherein said pharmaceutical composition isfor administration by a dosage regimen comprising at least two coursesof therapy, each course of therapy comprising in sequence a treatmentsession followed by an interval session of non-treatment.

In another aspect, the present invention provides method for treating adisease, disorder, condition or injury of the Central Nervous System(CNS) that does not include the autoimmune neuroinflammatory diseaserelapsing-remitting multiple sclerosis (RRMS), said method comprisingadministering to an individual in need thereof a pharmaceuticalcomposition according to any one of claims 1 to 24, wherein saidpharmaceutical composition is administered by a dosage regime comprisingat least two courses of therapy, each course of therapy comprising insequence a treatment session followed by an interval session of anon-treatment period.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B depict the choroid plexus (CP) activity along diseaseprogression in the 5XFAD transgenic mouse model of AD (AD-Tg). (A) mRNAexpression levels for the genes icam1, vcam1, cxcl10 and ccl2, measuredby RT-qPCR, in CPs isolated from 1, 2, 4 and 8-month old AD-Tg mice,shown as fold-change compared to age-matched WT controls (n=6-8 pergroup; Student's t test for each time point). (B) Representativemicroscopic images of CPs of 8-month old AD-Tg mice and age-matched WTcontrols, immunostained for the epithelial tight junction moleculeClaudin-1, Hoechst nuclear staining, and the integrin ligand, ICAM-1(scale bar, 50 μm). In all panels, error bars represent mean±s.e.m.; *,P<0.05; **, P<0.01;***, P<0.001.

FIGS. 2A-C show (A) Quantification of ICAM-1 immunoreactivity in humanpostmortem CP of young and aged non-CNS diseased, and AD patients (n=5per group; one-way ANOVA followed by Newman-Keuls post hoc analysis);(B) flow cytometry analysis for IFN-γ-expressing immune cells(intracellularly stained, and pre-gated on CD45) in CPs of 8-month oldAD-Tg mice and age-matched WT controls. Shaded histogram representsisotype control (n=4-6 per group; Student's t test); and (C) mRNAexpression levels of ifn-γ, measured by RT-qPCR, in CP tissues isolatedfrom 4- and 8-month old AD-Tg mice, compared to age-matched WT controls(n=5-8 per group; Student's t test for each time point). In all panels,error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 3A-B depict (A) representative flow cytometry plots of CD4⁺ Foxp3⁺splenocyte frequencies (pre-gated on TCRβ) in 8-month old AD-Tg and WTcontrol mice; and (B) quantitative analysis of splenocytes from 1, 2, 4and 8-month AD-Tg and WT control mice (n=6-8 per group; Student's t testfor each time point). In all panels, error bars represent mean±s.e.m.;*, P<0.05; **, P<0.01;***, P<0.001.

FIG. 4 shows gating strategy and representative flow cytometry plots ofsplenocytes from AD-Tg/Foxp3-DTR^(+/−) mice, 1 day after the lastinjection of DTx. DTx was injected i.p. for 4 constitutive days,achieving ˜99% depletion of Foxp3⁺ cells.

FIGS. 5A-G show the effects of transient depletion of Tregs in AD-Tgmice. (A) AD-Tg/Foxp3-DTR⁺ (which express the DTR transgene) and anon-DTR-expressing AD-Tg littermate (AD-Tg/Foxp3-DTR⁻) control groupwere treated with DTx for 4 constitutive days. CP mRNA expression levelsfor the genes icam1, cxcl10 and ccl2, measured by RT-qPCR, in 6-monthold DTx-treated AD-Tg mice. 1 day after the last DTx injection (n=6-8per group; Student's t test). (B-D) How cytometry analysis of the brainparenchyma (excluding the choroid plexus, which was separately excised)of 6-month old DTx-treated AD-Tg mice and controls, 3 weeks followingthe last DTx injection. Quantitative flow cytometry analysis showingincreased numbers of CD11b^(high)/CD45^(high) mo-MΦ and CD4⁺ T cells(B), and representative flow cytometry plots (C) and quantitativeanalysis (D) of CD4⁺Foxp3⁺ Treg frequencies, in the brain parenchyma ofAD-Tg/Foxp3-DTR⁺ mice and AD-Tg/Foxp3-DTR-controls treated with DTx(n=3-7 per group; Student's t test). (E) mRNA expression levels of foxp3and 00 in the brain parenchyma of 6-month old DTx-treated AD-TgAD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR-contros, 3 weeks after the last DTxinjection (n=6-8 per group; Student's t test). (F) quantitative analysisof GFAP immunostaining, showing reduced astrogliosis in hippocampalsections from 6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ andAD-Tg/Foxp3-DTR⁻ control mice, 3 weeks following the last DTx injection(scale bar, 50 μm; n=3-5 per group; Student's t test). (G) mRNAexpression levels of il-12p40 and tnf-α in the brain parenchyma, 3 weeksfollowing the last DTx injection (n=6-8 per group; Student's t test). Inall panels, error bars represent mean±s.e.m.; *, P <0.05; **,P<0.01;***, P<0.001.

FIGS. 6A-E show the effect of transient depletion of Tregs on Aβ plaqueslearning/memory performance. (A) Representative microscopic images and(B) quantitative analysis of the brains of 5-month old DTx-treatedAD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR⁻ control mice, 3 weeks after thelast DTx injection, immunostained for Aβ plaques and Hoechst nuclearstaining (scale bar, 250 μm). Mean Aβ plaque area and numbers in thehippocampal dentate gyrus (DG) and the 5^(th) layer of the cerebralcortex were quantified (in 6 μm brain slices; n=5-6 per group; Student'st test). FIGS. 6C-E) show Morris water maze (MWM) test performance of6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ and control mice, 3 weeks afterthe last DTx injection. Following transient Treg depletion, AD-Tg miceshowed better spatial learning/memory performance in the (C)acquisition, (D) probe and (E) reversal phases of the MWM, relative toAD-Tg controls (n=7-9 per group; two-way repeated measures ANOVAfollowed by Bonferroni post-hoc analysis for individual paircomparisons; *, P<0.05 for overall acquisition, probe, and reversal). Inall panels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***,P<0.001.

FIG. 7 shows mRNA expression levels of ifn-γ, measured by RT-qPCR, inCPs isolated from 6- and 12-month old APP/PS1 AD-Tg mice (a mouse modelfor Alzheimer's disease (see Materials and Methods)), compared toage-matched WT controls (n=5-8 per group; Student's t test). Error barsrepresent mean±s.e.m.; *, P<0.05.

FIGS. 8A-I show the therapeutic effect of administration of weeklyGlatiramer acetate (GA) in AD-Tg mice. (A) Schematic representation ofweekly-GA treatment regimen. Mice (5-month old) were s.c. injected withGA (100 μg), twice during the first week (on day 1 and 4), and onceevery week thereafter, for an overall period of 4 weeks. The mice wereexamined for cognitive performance, 1 week (MWM), 1 month (RAWM) and 2months (RAWM, using different experimental spatial settings) after thelast injection, and for hippocampal inflammation. FIGS. 8B-D show mRNAexpression levels of genes in the hippocampus of untreated AD-Tg mice,and AD-Tg mice treated with weekly-GA, at the age of 6 m, showing (B)reduced expression of pro-inflammatory cytokines such as TNF-α, IL-1βand IL-12p40, (C) elevation of the anti-inflammatory cytokines IL-10 andTGF-β, and of (D) the neurotropic factors, IGF-1 and BDNF, in weekly-GAtreated mice (n=6-8 per group; Student's t test). In FIGS. 8E-G, AD-Tgmice (5 months old) were treated with either weekly-GA or with vehicle(PBS), and compared to age-matched WT littermates in the MWM task at theage of 6 m. Treated mice showed better spatial learning/memoryperformance in the acquisition (E), probe (F) and reversal (G) phases ofthe MWM, relative to controls (n=6-9 per group; two-way repeatedmeasures ANOVA followed by Bonferroni post-hoc for individual paircomparisons). FIGS. 8H-I show cognitive performance of the same mice inthe RAWM task, 1 month (H) or 2 months (I) following the last GAinjection (n=6-9 per group; two-way repeated measures ANOVA followed byBonferroni post-hoc for individual pair comparisons). Data arerepresentative of at least three independent experiments. In all panels,error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 9A-H show further therapeutic effects of administration ofweekly-GA in AD-Tg mice. A-B shows 5XFAD AD-Tg mice that were treatedwith either weekly-GA, or vehicle (PBS), and were examined at the end ofthe 1^(st) week of the administration regimen (after a total of two GAinjections). Flow cytometry analysis for CD4⁺Foxp3⁺ splenocytefrequencies (A), and CP IFN-γ-expressing immune cells (B;intracellularly stained and pre-gated on CD45), in treated 6-month oldAD-Tg mice, compared to age-matched WT controls (n=4-6 per group;one-way ANOVA followed by Newman-Keuls post hoc analysis). (C) mRNAexpression levels for the genes icam1, cxcl10 and ccl2, measured byRT-qPCR, in CPs of 4-month old AD-Tg mice, treated with either weekly-GAor vehicle, and examined either at the end of the 1^(st) or 4^(th) weekof the weekly-GA regimen (n=6-8 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). FIGS. 9D-E show representative imagesof brain sections from 6-month old AD-Tg/CX₃CR1^(GFP/+) BM chimerasfollowing weekly-GA. CX₃CR1^(GFP) cells were localized at the CP of thethird ventricle (3V; i), the adjacent ventricular spaces (ii), and theCP of the lateral ventricles (LV; iii) in AD-Tg mice treated withweekly-GA (D; scale bar, 25 μm). Representative orthogonal projectionsof confocal z-axis stacks, showing co-localization of GFP⁺ cells withthe myeloid marker, CD68, in the CP of 7-month old AD-Tg/CX₃CR1^(GFP/+)mice treated with weekly-GA, but not in control PBS-treatedAD-Tg/CX₃CR1^(GFP/+) mice (E; scale bar, 25 μm). (F) CX₃CR1^(GFP) cellsare co-localized with the myeloid marker IBA-1 in brains of GA-treatedAD-Tg/CX₃CR1^(GFP/+) mice in the vicinity of Aβ plaques, andco-expressing the myeloid marker, IBA-1 (scale bar, 25 μm). FIGS. 9G-Hshow representative flow cytometry plots of cells isolated from thehippocampus of 4-month old WT, untreated AD-Tg, and AD-Tg mice, on the2^(nd) week of the weekly-GA regimen. CD11b^(high)/CD45^(high) mo-MΦwere gated (G) and quantified (H; n=4-5 per group; one-way ANOVAfollowed by Newman-Keuls post hoc analysis). In all panels, error barsrepresent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 10A-H depict the therapeutic effect of administration of a p300inhibitor (C646) in AD-Tg mice. In FIGS. 10A-B, aged mice (18 months)were treated with either p300i or vehicle (DMSO) for a period of 1 week,and examined a day after cessation of treatment. Representative flowcytometry plots showing elevation in the frequencies of CD4⁺ T cellsexpressing IFN-γ in the spleen (A), and IFN-γ-expressing immune cellnumbers in the CP (B), following p300i treatment. FIGS. 10C-E showrepresentative microscopic images (C), and quantitative analysis, of Aβplaque burden in the brains of 10-month old AD-Tg mice, which receivedeither p300i or vehicle (DMSO) for a period of 1 week, and weresubsequently examined after 3 additional weeks. Brains wereimmunostained for Aβ plaques and by Hoechst nuclear staining (n=5 pergroup; Scale bar, 250 μm). Mean Aβ plaque area and plaque numbers werequantified in the hippocampal DG (D) and the 5^(th) layer of thecerebral cortex (E) (in 6 μm brain slices; n=5-6 per group; Student's ttest). (F) Schematic representation of the p300i treatment (or DMSO asvehicle) administration regimen to the different groups of AD-Tg mice atthe age of 7 months, in either 1 or 2 sessions. FIGS. 10G-H show thechange mean of Aβ plaque percentage coverage of the cerebral cortex(5^(th) layer) (G), and the change in mean cerebral soluble Aβ₁₄₀ andAβ₁₄₂ protein levels (H), relative to the untreated AD-Tg group (Aβ₁₄₀and Aβ₁₄₂ mean level in untreated group, 90.5±11.2 and 63.8±6.8 pg/mgtotal portion, respectively; n=5-6 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). In all panels, error bars representmean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 11A-B show the therapeutic effect of administration of anti-PD1antibody in AD-Tg mice. (A) Schematic representation of the experimentalgroups of mice, their age, treatment administration regimens, and thetime point in which mice were examined. At 10 months of age, 5XFADAlzheimer's' disease (AD) transgenic (Tg) mice were injected i.p. witheither 250 μg of anti-PD1 (RMP1-14) or control IgG (rat) antibodies, onday 1 and day 4 of the experiment, and were examined 3 weeks after fortheir cognitive performance by radial arm water maze (RAWM) spatiallearning and memory task. Age matched untreated WT and AD-Tg mice wereused as controls. (B) show cognitive performance, as assessed by radialarm water maze (RAWM) spatial learning and memory task. Data wereanalyzed using two-way repeated-measures ANOVA, and Bonferroni post-hocprocedure was used for follow-up pairwise comparison. n=6-12 per group.In all panels, error bars represent mean±s.e.m.; *, P<0.05; **,P<0.01;***, P<0.001.

FIGS. 12A-B show the systemic effect on IFN-γ⁺ producing T cells ofadministration of anti-PD1 antibody in AD-Tg mice. (A) Schematicrepresentation of the experimental groups of mice, their age, treatmentadministration regimens, and the time point in which mice were examined.Mice were injected i.p. with either 250 μg of anti-PD1 (RMP1-14) orcontrol IgG (rat) antibodies, on day 1 and day 4 of the experiment, andexamined on day 7. (B) Flow cytometry analysis for CD4⁺IFN-γ⁺ T cellsplenocyte frequencies (intracellularly stained and pre-gated on CD45and TCR-β), in PD-1 or IgG treated AD-Tg mice, and untreated AD-Tg andWT controls (n=4-6 per group; one-way ANOVA followed by Newman-Keulspost hoc analysis; **, P<0.01 between the indicted treated groups; errorbars represent mean±s.e.m.).

FIGS. 13A-B show the effect on the CP following anti-PD1 treatment inAD-Tg mice. AD-Tg mice at the age of 10 months were either treated withPD-1, IgG, or left untreated. Mice were injected i.p. with either 250 μgof anti-PD1 (RMP1-14) or control IgG (rat) antibodies, on day 1 and day4 of the experiment, and examined on day 7. (A) CP IFN-γ levels, asmeasured by real-time quantitative PCR (RT-qPCR), positively correlated(Pearson's r=0.6284, P<0.05), to and negatively correlated to CD4⁺IFN-γ⁺T cell splenocyte frequencies, as measured by flow cytometry. Anopposite, negative trend was observed in the same mice, when CP IFN-γlevels were compared to CD4⁺Foxp3⁺CD25⁺ Tregs splenocyte frequencies(n=3-4 per group). (B) mRNA expression levels for the genes cxcl10 andccl2, measured by RT-qPCR, in CPs of the same mice (n=3-4 per group;one-way ANOVA followed by Newman-Keuls post hoc analysis). In allpanels, error bars represent mean±s.e.m.; *, P<0.05.

FIGS. 14A-B show the therapeutic effect of administration of anti-PD1antibody in AD-Tg mice, when comparing one vs. two courses of treatment.Half of the mice in the group of mice described in FIG. 11A-B whichreceived one course of anti-PD1 treatment, either received anothercourse of anti-PD1 treatment following the first RAWM task, or leftuntreated. Following additional 3 weeks, all mice were tested by RAWMusing different and new experimental settings of spatial cues forcognitive learning and memory. (A) Schematic representation of theexperimental groups of mice, their age, treatment administrationregimens, and the time point in which mice were examined. For eachcourse of treatment, mice were injected i.p. with either 250 ug ofanti-PD1 (RMP1-14) or control IgG (rat) antibodies. (B) show cognitiveperformance, as assessed by RAWM spatial learning and memory task. Datawere analyzed using two-way repeated-measures ANOVA, and Bonferronipost-hoc procedure was used for follow-up pairwise comparison. n=6-12per group. In all panels, error bars represent mean±s.e.m.; *, P<0.05;**, P<0.01;***, P<0.001.

FIGS. 15A-H show the adverse effect on AD pathology of systemic Treglevels augmented by all-trans retinoic acid (ATRA). FIGS. 15A-B showrepresentative flow cytometry plots (A), and quantitative analysis (B),showing elevation in frequencies of CD4⁺/Foxp3⁺/CD25⁺ Treg splenocytesin 5-month old AD-Tg mice, which received either ATRA or vehicle (DMSO)for a period of 1 week (n=5 per group; Student's t test). FIGS. 15C-Fshow representative microscopic images (C), and quantitative analysis(D, E, F), of Aβ plaque burden and astrogliosis in the brains of AD-Tgmice, which at the age of 5-months were treated with either ATRA orvehicle (DMSO) for a period of 1 week, and subsequently examined after 3additional weeks. Brains were immunostained for Aβ plaques, GFAP(marking astrogliosis), and by Hoechst nuclear staining (n=4-5 pergroup; Scale bar, 250 μm). Mean Aβ plaque area and plaque numbers werequantified in the hippocampal DG and the 5⁺ layer of the cerebralcortex, and GFAP immunoreactivity was measured in the hippocampus (in 6μm brain slices; n=5-6 per group; Student's t test). (G) Levels ofsoluble Aβ₁₋₄₀ and Aβ₁₋₄₂, quantified by ELISA, in the cerebral brainparenchyma AD-Tg mice, which at the age of 5-months were treated witheither ATRA or vehicle (DMSO) for a period of 1 week, and subsequentlyexamined after 3 additional weeks (n=5-6 per group; Student's t test).(H) Cognitive performance in the RAWM task of AD-Tg mice which at theage of 5-months were treated with either ATRA or vehicle (DMSO) for aperiod of 1 week, and subsequently examined after 3 additional weeks(n=5 per group; two-way repeated measures ANOVA followed by Bonferronipost-hoc for individual pair comparisons). In all panels, error barsrepresent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 16A-F show the adverse effect on AD pathology of systemic Treglevels augmented by weekly-GA administration. (A) Schematicrepresentation of daily-GA treatment regimen compared to the weekly-GAregimen. In the daily-GA treated group, mice were s.c. injected dailywith 100 μg of GA for a period of 1 month. (B) Cognitive performance ofdaily-GA and weekly-GA treated 7-month old AD-Tg mice, compared toage-matched WT and untreated AD-Tg mice, as assessed by the averagenumbers of errors per day in the RAWM learning and memory task (n=6-8per group; one-way ANOVA followed by Newman-Keuls post hoc analysis).(C) Representative microscopic images of the cerebral cortex and thehippocampus (HC) of untreated AD-Tg, and daily or weekly-GA treatedAD-Tg mice, immunostained for Aβ plaques and for Hoechst nuclearstaining (scale bar, 250 μm). FIGS. 16D-F show quantification of Aβplaque size and numbers (per 6 μm slices) in GA treated (daily-GA andweekly-GA groups) and untreated AD-Tg mice. Weekly-GA treated AD-Tg miceshowed reduction in Aβ plaque load as a percentage of the total area oftheir hippocampal dentate gyrus (DG), and in mean Aβ plaque numbers (n=6per group; one-way ANOVA followed by Newman-Keuls post hoc analysis). Inall panels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***,P<0.001.

DETAILED DESCRIPTION

It has been found in accordance with the present invention that ashort-term transient depletion of Foxp3⁺ regulatory T cells (Tregs) in amouse model of Alzheimer's disease (AD-Tg mice) results in improvedrecruitment of leukocytes to the CNS through the brain's choroid plexus,elevated numbers of CNS-infiltrating anti-inflammatory monocyte-derivedmacrophages mo-MΦ and CD4⁺ T cells, and a marked enrichment of Foxp3⁺Tregs that accumulates within the brain. Furthermore, the long-termeffect of a single session of treatment lead to a reduction inhippocampal gliosis and reduced mRNA expression levels ofpro-inflammatory cytokines within the brain. Importantly, the effect ondisease pathology includes reduced cerebral amyloid beta (Aβ) plaqueburden in the hippocampal dentate gyrus, and the cerebral cortex (5^(th)layer), two brain regions exhibiting robust Aβ plaque pathology in theAD-Tg mice. Most importantly, the short-term transient depletion ofTregs is followed by a dramatic improvement in spatial learning andmemory, reaching cognitive performance similar to that of wild type mice(Examples 2 and 3). Taken together, these findings demonstrate that ashort session of Treg depletion, followed by a period of nointervention, results in transiently breaking Treg-mediated systemicimmune suppression in AD-Tg mice, which enables recruitment ofinflammation-resolving cells, mo-MΦ and Tregs, to the brain, and lead toresolution of the neuroinflammatory response, clearance of AP, andreversal of cognitive decline. These findings strongly argue against thecommon wisdom in this field of research, according to which increasingsystemic immune suppression would result in mitigation of theneuroinflammatory response. On the contrary, our findings show thatboosting of the systemic response, by a short-term, brief and transient,reduction in systemic Treg-mediated suppression, is needed in order toachieve inflammation-resolving immune cell accumulation, including Tregsthemselves, within the brain, thus fighting off AD pathology.

The specificity of the inventors approach presented herein has beensubstantiated by using several independent experimental paradigms, asdetailed below. Briefly, first the inventors used an immunomodulatorycompound in two different administration regimens that led to oppositeeffects on peripheral Treg levels, on CP activation, and on diseasepathology; a daily administration regimen that augments peripheral Treglevels (Weber et al, 2007), and a weekly administration regimen, whichthey found to reduce peripheral Treg levels (Example 3 and Example 5).The inventors also provide a direct functional linkage betweenperipheral Treg levels and disease pathology when demonstrating in AD-Tgmice, by either transient in vivo genetic depletion of Tregs (Example2), or by pharmacologic inhibition of their Foxp3 function (Examples 3and 4), that these manipulations result in activation of the CP forfacilitating leukocyte trafficking to the CNS, inflammation-resolvingimmune cell accumulation at sites of pathology, clearance of cerebral Aβplaques, and skewing of the immunological milieu of the brain parenchymatowards the resolution of inflammation.

It has further been found in accordance with the present invention thatinfrequent administration of a universal antigen, Copolymer-1, for alimited period of time (representing one session of treatment) reducesTreg-mediated systemic immune suppression, and improves selectiveinfiltration of leukocytes into the CNS by increasing the brain'schoroid plexus gateway activity, leading to dramatic beneficial effectin Alzheimer's disease pathology (Example 3), while daily administrationof Copolymer 1, that enhance Treg immune suppression (Hong et al, 2005;Weber et al, 2007), showed no beneficial effect, or even some modestdetrimental effect, on disease pathology (Example 5). The inventors ofthe present invention further show herein that direct interference withFoxp3 Treg activity, either by inhibition of p300 with a specific smallmolecule inhibitor (p300i), or interaction with the PD-1 receptor by ananti-PD-1 antibody, improves choroid plexus gateway activity in AD-Tgmice, and mitigates Alzheimer's disease pathology (Example 4).

Importantly, each of these examples provided by the inventors,demonstrate a different intervention which causes short term reductionin systemic immune suppression: Copolymer-1 acts as an immunomodulatorycompound, p300i as a small molecule which decreases Foxp3 acetylationand Treg function, and anti-PD-1 is used as a neutralizing antibody forPD-1 expressed on Tregs. These therapeutic approaches were used for ashort session of treatment that transiently augmented immune response inthe periphery, mainly by elevation of peripheral IFN-γ levels andIFN-γ-producing cells, thus activating the brain's choroid plexusallowing selective infiltration of T cells and monocytes into the CNS,and homing of these cells to sites of pathology and neuroinflammation.It was also found herein that repeated sessions of treatment interruptedby interval sessions of non-treatment dramatically improve the efficacyof the treatment relative to a single session of treatment (Example 4).The following time interval of non-treatment allowed transientaugmentation in Treg levels and activities within the brain,facilitating the resolution of neuroinflammation, and inducingenvironmental conditions in favor of CNS healing and repair,subsequently leading to tissue recovery. In each of these cases theeffect on brain pathology was robust, involving the resolution of theneuroinflammatory response, amyloid beta plaque clearance from AD micebrains, and reversal of cognitive decline. The specificity of thecurrent approach has further been substantiated using a genetic model oftransient depletion of Foxp3⁺ regulatory T cells, in transgenic mousemodel of AD (Example 2).

Thus, it has been found in accordance with the present invention thatsystemic Foxp3⁺CD4⁺ Treg-mediated immunosuppression interferes withability to fight off AD pathology, acting at least in part, byinhibiting IFN-γ-dependent activation of the CP, needed fororchestrating recruitment of inflammation-resolving leukocytes to theCNS (Schwartz & Baruch, 2014b). Systemic Tregs are crucial formaintenance of autoimmune homeostasis and protection from autoimmunediseases (Kim et al, 2007). However, our findings suggest that underneurodegenerative conditions, when a reparative immune response isneeded in the brain, the ability to mount this response is interferedwith by systemic Tregs. Nevertheless according to our results, Tregs areneeded within the brain, home to sites of neuropathology, and performlocally an anti-inflammatory activity. The present invention representsa unique and unexpected solution for the apparent contradictory needs infighting off progressive neuronal death as in AD; transientlyreducing/inhibiting Tregs in the circulation on behalf of increasingTregs in the diseased brain. Hence, a short-term and transient reductionin peripheral immune suppression, which allows the recruitment ofanti-inflammatory cells, including Tregs and mo-MΦ, to sites of cerebralplaques, leads to a long-term effect on pathology. Notably, however, atransient reduction of systemic Treg levels or activities may contributeto disease mitigation via additional mechanisms, including supporting aCNS-specific protective autoimmune response (Schwartz & Baruch, 2014a),or augmenting the levels of circulating monocytes that play a role inclearance of vascular Aβ (Michaud et al, 2013).

Though neurodegenerative diseases of different etiology, share a commonlocal neuroinflammatory component, our results strongly argue againstsimplistic characterization of all CNS pathologies as diseases thatwould uniformly benefit from systemic anti-inflammatory therapy. Thus,while autoimmune inflammatory brain pathologies, such asRelapsing-Remitting Multiple Sclerosis (RRMS), benefit from continuoussystemic administration of anti-inflammatory and immune-suppressivedrugs to achieve long lasting peripheral immune suppression, it willeither be ineffective or detrimentally affect (Example 5) pathology inchronic neurodegenerative diseases such as in the case of AD, primaryprogressive multiple sclerosis (PP-MS) and secondary-progressivemultiple sclerosis (SP-MS). Moreover, our findings shed light on themisperception regarding the role of systemic vs. tissue-associated Tregsin these pathologies (He & Balling, 2013). Since the immune-brain axisis part of life-long brain plasticity (Baruch et al, 2014), andneurodegenerative diseases are predominantly age-related, our presentfindings also point to a more general phenomenon, in which systemicimmune suppression interferes with brain function. Accordingly,short-term periodic courses of reducing systemic immune suppression mayrepresent a therapeutic or even preventive approach, applicable to awide range of brain pathologies, including AD and age-associateddementia.

Importantly, the inventors approach and findings present herein in ADmouse models, do not directly target any disease-specific factor in AD,such as amyloid beta or tau pathology, but rather demonstrate a novelapproach which is expected to be clinically applicable in a wide rangeof CNS pathologies—transient reduction of systemic Treg-mediated immunesuppression in order to augment recruitment of inflammation-resolvingimmune cells to sites of pathology within the CNS.

In view of the unexpected results described above, the present inventionprovides a pharmaceutical composition comprising an active agent thatcauses reduction of the level of systemic immunosuppression in anindividual for use in treating a disease, disorder, condition or injuryof the CNS that does not include the autoimmune neuroinflammatorydisease, relapsing-remitting multiple sclerosis (RRMS), wherein saidpharmaceutical composition is for administration by a dosage regimencomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session followed by an intervalsession of non-treatment.

In certain embodiments, the dosage regimen is calibrated such that thelevel of systemic immunosuppression is transiently reduced.

The term “treating” as used herein refers to means of obtaining adesired physiological effect. The effect may be therapeutic in terms ofpartially or completely curing a disease and/or symptoms attributed tothe disease. The term refers to inhibiting the disease, i.e. arrestingor slowing its development; or ameliorating the disease, i.e. causingregression of the disease.

The term “non-treatment session” is used interchangeably herein with theterm “period of no treatment” and refers to a session during which noactive agent is administered to the individual being treated.

The term “systemic presence” of regulatory T cells as used herein refersto the presence of the regulatory T cells (as measured by their level oractivity) in the circulating immune system, i.e. the blood, spleen andlymph nodes. It is a well-known fact in the field of immunology that thecell population profile in the spleen is reflected in the cellpopulation profile in the blood (Zhao et al, 2007).

The present treatment is applicable to both patients that show elevationof systemic immune suppression, as well as to patients that do not showsuch an elevation. Sometimes the individual in need for the treatmentaccording to the present invention has a certain level of peripheralimmunosuppression, which is reflected by elevated frequencies or numbersof Tregs in the circulation, and/or their enhanced functional activityand/or a decrease in IFNγ-producing leukocytes and/or decreasedproliferation of leukocytes in response to stimulation. The elevation offrequencies or numbers of Tregs can be in total numbers or as percentageof the total CD4 cells. For example, it has been found in accordancewith the present invention that an animal model of Alzheimer's diseasehas higher frequencies of Foxp3 out of CD4 cells as compared withwild-type mice. However, even if the levels of systemic Treg cells isnot elevated, their functional activity is not enhanced, the level ofIFNγ-producing leukocytes is not reduced or the proliferation ofleukocytes in response to stimulation is not decreased, in saidindividual, the method of the present invention that reduces the levelor activity of systemic immunosuppression is effective in treatingdisease, disorder, condition or injury of the CNS that does not includethe autoimmune neuroinflammatory disease RRMS. Importantly, saidsystemic immune suppression can also involve additional immune celltypes except of Tregs, such as myeloid-derived suppressor cells (MDSCs)(Gabrilovich & Nagaraj, 2009).

The level of systemic immunosuppression may be detected by variousmethods that are well known to those of ordinary skill in the art. Forexample, the level of Tregs may be measured by flow cytometry analysisof peripheral blood mononuclear cells or T lymphocytes, immunostainedeither for cellular surface markers or nuclear intracellular markers ofTreg (Chen & Oppenheim, 2011), CD45, TCR-β, or CD4 markers oflymphocytes, and measuring the amount of antibody specifically bound tothe cells. The functional activity of Tregs may be measured by variousassays; For example the thymidine incorporation assay is being commonlyused, in which suppression of anti-CD3 mAb stimulated proliferation ofCD4⁺CD25⁻ T cells (conventional T cells) is measured by [³H]thymidineincorporation or by using CFSE (5-(and 6)-carboxyfluorescein diacetatesuccinimidyl ester, which is capable of entering the cells; celldivision is measured as successive halving of the fluorescence intensityof CFSE). The number of IFNγ-producing leukocytes or their activity ortheir proliferation capacity can easily be assessed by a skilled artisanusing methods known in the art; For example, the level of IFNγ-producingleukocytes may be measured by flow cytometry analysis of peripheralblood mononuclear cells, following short ex-vivo stimulation andgolgi-stop, and immunostaining by IFNγ intracellular staining (usinge.g., BD Biosciences Cytofix/cytoperm™ fixation/permeabilization kit),by collecting the condition media of these cells and quantifying thelevel of secreted cytokines using ELISA, or by comparing the ratio ofdifferent cytokines in the condition media, for example IL2/IL10,IL2/IL4, INFγ/TGFβ, etc. The levels of MDSCs in the human peripheralblood easily can be assessed by a skilled artisan, for example by usingflow cytometry analysis of frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+,DR⁻/LIN⁻/CD33+ and DR(−/low)/CD14+ cells, as described (Kotsakis et al,2012).

In humans, the peripheral/systemic immunosuppression may be consideredelevated when the total number of Tregs in the circulation is higherthan 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more than in ahealthy control population, the percentage of Treg cells out of thetotal CD4+ cells is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population, or the functionalactivity of Tregs is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population. Alternatively, theperipheral/systemic immunosuppression may be considered elevated whenthe level of IFNγ-producing leukocytes or their activity is reducedrelative to that of a healthy control population by 10, 20, 30, 40, 50,60, 70, 80, 90 or 100%; or the proliferation of leukocytes in responseto stimulation is reduced relative to that of a healthy controlpopulation by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

An agent may be considered an agent that causes reduction of the levelof systemic immunosuppression when, upon administration of the agent toan individual, the total number of Tregs in the circulation of thisindividual is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% ascompared with the level before administration of the agent, thepercentage of Treg cells out of the total CD4+ cells drops by 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% relative to that of a healthy controlpopulation or the functional activity of Tregs is reduced by 10, 20, 30,40, 50, 60, 70, 80, 90 or 100% as compared with the level beforeadministration of the agent. Alternatively, an agent may be consideredan agent that causes reduction of the level of systemicimmunosuppression when, upon administration of the agent to anindividual, the total number of IFNγ-producing leukocytes or theiractivity is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%; orthe proliferation of leukocytes in response to stimulation is increasedrelative to that of a healthy control population by 10, 20, 30, 40, 50,60, 70, 80, 90 or 100%.

The agent used according to the present invention may be any agent thatdown-regulates the level or activity of regulatory T cells or interferewith their activity, but may alternatively be limited to a group of suchagents excluding an agent selected from the group consisting of: (i)dopamine or a pharmaceutically acceptable salt thereof; (ii) a dopamineprecursor or a pharmaceutically acceptable salt thereof; (iii) anagonist of the dopamine receptor type 1 family (D1-R agonist) or apharmaceutically acceptable salt thereof; and (iv) an antagonist of thedopamine receptor type 2 family (D2-R antagonist) or a pharmaceuticallyacceptable salt thereof, even though these agents are not know for useaccording to the course of therapy according to the present invention.

In certain embodiments, the treatment session comprises administeringthe pharmaceutical composition to the individual and the treatmentsession is maintained at least until the level falls below a reference,the administering is paused during the interval session, and theinterval session is maintained as long as the level is below thereference. The reference may be selected from (a) the level of systemicpresence or activity of regulatory T cells or myeloid-derived suppressorcells measured in the most recent blood sample obtained from saidindividual before said administering; or (b) the level of systemicpresence or activity of regulatory T cells or myeloid-derived suppressorcells characteristic of a population of individuals afflicted with adisease, disorder, condition or injury of the CNS.

Alternatively, the treatment session comprises administering thepharmaceutical composition to the individual and the treatment sessionis maintained at least until the systemic presence or level ofIFNγ-producing leukocytes, or the rate of proliferation of leukocytes inresponse to stimulation rises above a reference, the administering ispaused during the interval session, and the interval session ismaintained as long as said level is above said reference, wherein thereference is selected from (a) the level of systemic presence oractivity of IFNγ-producing leukocytes, or the rate of proliferation ofleukocytes in response to stimulation, measured in the most recent bloodsample obtained from said individual before said administering; or (b)the level of systemic presence or activity of IFNγ-producing leukocytes,or the rate of proliferation of leukocytes in response to stimulation,characteristic of a population of individuals afflicted with a disease,disorder, condition or injury of the CNS.

The length of the treatment and interval sessions may be determined byphysicians in clinical trials directed to a certain patient populationand then applied consistently to this patient population, without theneed for monitoring the level of immunosuppression on a personal basis.

In certain embodiments, the treatment session may be between 3 days andfour weeks long, for example between one and four weeks long.

In certain embodiments, the interval session may be between one week andsix months, for example between two weeks and six months long, inparticular between 3 weeks and six months long.

In the treatments session, the administration of the pharmaceuticalcomposition may be repeated administration, for example thepharmaceutical composition may be administered daily, or once every two,three, four, five or six days, once weekly, once every two weeks, onceevery three weeks or once every four weeks. These frequencies areapplicable to any active agent, may be based on commonly used practicesin the art, and may finally be determined by physicians in clinicaltrials. Alternatively, the frequency of the repeated administration inthe treatment session could be adapted according to the nature of theactive agent, wherein for example, a small molecule may be administereddaily; an antibody may be administered once every 3 days; and copolymer1 is administered weekly, once every two weeks, once every three weeksor once every four weeks. It should be understood that when an agent,such as copolymer 1, is administered during a treatment session at arelatively low frequency, for example once per week during a treatmentsession of one month, or once per month during a treatment session ofsix months, this treatment session is followed by a non-treatmentinterval session, the length of which is longer than the period betweenthe repeated administrations during the treatment session (i.e. longerthan one week or one month, respectively, in this example). The pause ofone week or one month between the administrations during the treatmentsession in this example is not considered an interval session.

The lengths of the treatment session and the interval session may beadjusted to the frequency of the administration such that, for example,a frequency of administering the active agent once every 3 days mayresult in a treatment session of 6 or 9 days and an interval sessionthat is commenced accordingly.

As an alternative to a predetermined general treatment regiment, thelevel of immunosuppression may be calibrated to a desired level for eachpatient who is being treated (personalized medicine), by monitoring thelevel or activity of Treg cells (or IFN-γ-producing leukocytes orproliferation rate of leukocytes in response to stimulation)individually, and adjusting the treatment session, the frequency ofadministration and the interval session empirically and personally asdetermined from the results of the monitoring.

Thus, the length of the treatment session may be determined by (a)monitoring the level of systemic presence or activity of regulatory Tcells in the individual by measuring the level in a blood sampleobtained from the individual within a predetermined time-periodfollowing said administering; (b) comparing the level measured in (a)with the reference mentioned above and determining whether the level isdifferent from the reference; (c) deciding, based on the relation ofsaid level measured in (a) to said reference, whether to continue thetreatment session by repeating the administering or starting the nextinterval session by refraining from repeating the administration; and(d) repeating the administering or starting the next interval sessionaccording to the decision in (c). Alternatively, the level ofIFN-γ-producing leukocytes or proliferation rate of leukocytes inresponse to stimulation may be monitored and compared with anappropriate reference as mentioned above.

Similarly, the length of the interval session may be determined by (a)monitoring the level of systemic presence or activity of regulatory Tcells in the individual by measuring the level in a blood sampleobtained from the individual within a predetermined time-periodfollowing said administering; (b) comparing the level measured in (a)with the reference mentioned above and determining whether the level isdifferent from the reference; (c) deciding, based on the relation ofsaid level measured in (a) to said reference, whether to start a newcourse of therapy by repeating the administering and steps (a) and (b)or to prolong the interval session by repeating only steps (a) and (b);and (d) repeating the administering and steps (a) and (b) or only steps(a) and (b) according to the decision in (c). Alternatively, the levelof IFNγ-producing leukocytes or proliferation rate of leukocytes inresponse to stimulation may be monitored and compared with anappropriate reference as mentioned above.

In any case, the dosage regimen, i.e. the length of the treatmentsession and the interval session, is calibrated such that the reductionin the level of immunosuppression, for example as measured by areduction in the level of systemic presence or activity of regulatory Tcells in the individual, is transient.

In certain embodiments, the predetermined time-period, i.e. the timepassed between the most recent administration of the active agent andthe monitoring step, is between 2 days and six months.

In certain embodiments, the regulatory T cells that are monitored areCD4+ cells selected from FoxP3⁺ cells expressing one or more of CD25,CD127, GITR, CTLA-4 or PD-1; or FoxP3⁻ cells expressing one or more ofCD25, CD127, GITR, CTLA-4 or PD-1 surface molecules. In particular, acommon phenotype of regulatory T cells is CD4⁺CD25⁺FoxP3⁺ cells orCD4⁺CD25⁺FoxP3⁻ cells.

Agents capable of reducing the level of regulatory T cells are known inthe art (Colombo & Piconese, 2007) and these agents can be used inaccordance with the present invention. Each one of the citedpublications below is incorporated by reference as if fully disclosedherein.

Thus, the agent may be selected from, but is not necessarily limited to:(i) an antibody such as: (a) anti-PD-1, (b) anti-PD-L1 (c) anti-PD-L2(Coyne & Gulley, 2014; Duraiswamy et al, 2014; Zeng et al, 2013); (d)anti-CTLA-4 (Simpson et al, 2013; Terme et al, 2012); (e) anti-PD-1 incombination with interferon α (Terawaki et al, 2011); (f) anti-PD-1 incombination with anti-CTLA4; (g) anti-CD47 (Tseng et al, 2013); (h)anti-OX40 (Voo et al, 2013); (i) anti-VEGF-A (bevacizumab) (Terme et al,2013); (j) anti-CD25 (Zhou et al, 2013); (k) anti-GITR (GITR triggeringmAb (DTA-1) (Colombo & Piconese, 2007); (l) anti-CCR4; (m)anti-TIM-3/Galectin9 (Ju et al, 2014); (n) anti-killer-cellimmunoglobulin-like receptors (KIR); (o) anti-LAG-3; or (p) anti-4-1BB(ii) any combination of (a) to (p); (iii) any combination of (a) to (p)in combination with an adjuvant, for example anti-CTLA-4 antibody incombination with anti OX40 antibody and a TLR9 ligand such as CpG(Marabelle et al, 2013); (iv) a small molecule selected from: (a) A p300inhibitor (Liu et al, 2013), such as gemcitabine (low dose) (Shevchenkoet al, 2013), or C646 or analogs thereof, i.e. a compound of the formulaI:

wherein

R₁ is selected from H, —CO₂R₆, —CONR₆R₇, —SO₃H, or —SO₂NR₆R₇;

R₂ is selected from H, —CO₂R₆, or halogen, preferably Cl;

R₃ is selected from halogen, preferably F, —NO₂, —CN, —CO₂R₆, preferablyCO₂CH₃ or CO₂CH₂CH₃, or —CH₂OH;

R₄ and R₅ each independently is H or —C₁-C₆ alkyl, preferably methyl;

R₆ is H or —C₁-C₆ alkyl, preferably H, methyl or ethyl; and

R₇ is H or —C₁-C₆ alkyl, preferably H or methyl [see (Bowers et al,2010)];

(b) Sunitinib (Terme et al, 2012); (c) Polyoxometalate-1 (POM-1)(Ghiringhelli et al, 2012); (d) α,β-methyleneadenosine 5′-diphosphate(APCP) (Ghiringhelli et al, 2012); (e) arsenic trioxide (As₂O₃)(Thomas-Schoemann et al, 2012); (f) GX15-070 (Obatoclax) (Kim et al,2014); (g) a retinoic acid antagonist such as Ro 41-5253 (a syntheticretinoid and selective small molecule antagonist) (Galvin et al, 2013)or LE-135 (Bal et al, 2009); (h) an SIRPα (CD47) antagonist, such asCV1-hIgG4 (SIRPα variant) as sole agent or in combination with anti-CD47antibody (Weiskopf et al, 2013); (i) a CCR4 antagonist, such asAF399/420/18025 as sole agent or in combination with anti-CCR4 antibody(Pere et al, 2011); (j) an adenosin A2B receptor antagonist, such asPSB603 (Nakatsukasa et al, 2011); (k) an antagonist ofindoleamine-2,3-dioxygenase (IDO); or (l) an HIF-1 regulator; (iv) aprotein selected from: (a) Neem leaf glycoprotein (NLGP) (Roy et al,2013); or (b) sCTLA-4 (soluble isoform of CTLA-4) (Ward et al, 2013);(vi) a silencing molecule such as miR-126 antisense (Qin et al, 2013)and anti-galectin-1 (Gal-1) (Dalotto-Moreno et al, 2013); (vii) OK-432(lyophilized preparation of Streptococcus pyogenes) (Hirayama et al,2013); (viii) a combination of IL-12 and anti-CTLA-4; (ix) Copolymer 1or a copolymer that modulates Treg activity or level; (x) an antibioticagent, such as vancomycin (Brestoff & Artis, 2013; Smith et al, 2013) or(xi) any combination of (i) to (x).

In certain embodiments, the agent is an anti-PD-1 antibody, i.e. anantibody specific for PD-1.

Many anti-PD-1 antibodies are known in the art. For example, theanti-PD-1 antibody used in accordance with the present invention may beselected from those disclosed in Ohaegbulam et al. (Ohaegbulam et al,2015), the entire contents of which being hereby incorporated herein byreference, i.e. CT-011 (pidilizumab; Humanized IgG1; Curetech), MK-3475(lambrolizumab, pembrolizumab; Humanized IgG4; Merck), BMS-936558(nivolumab; Human IgG4; Bristol-Myers Squibb), AMP-224 (PD-L2 IgG2afusion protein; Astra7eneca), BMS-936559 (Human IgG4; Bristol-MyersSquibb), MEDI4736 (Humanized IgG; AstraZeneca), MPDL3280A (Human IgG;Genentech), MSB0010718C (Human IgG1; Merck-Serono); or the antibody usedin accordance with the present invention may be MEDI0680 (AMP-514;AstraZeneca) a humanized IgG4 mAb.

In certain embodiments, the CT-011 antibody may be administered to ahuman at a dosage of 0.2-6 mg/kg or between 1.5-6 mg/kg; the MK-3475antibody may be administered to a human at a dosage of 1-10 mg/kg;BMS-936558 may be administered to a human at a dosage of 0.3-20 mg/kg,0.3-10 mg/kg, 1-10 mg/kg or at 1 or 3 mg/kg; BMS-936559 may beadministered to a human at a dosage of 0.3-10 mg/kg; MPDL3280A may beadministered to a human at a dosage of 1-20 mg/kg; MEDI4736 may beadministered to a human at a dosage of 0.1-15 mg/kg; and MSB0010718C maybe administered to a human at a dosage of 1-20 mg/kg.

The anti-CTLA4 antibody may be Tremelimumab (Pfizer), a fully human IgG2monoclonal antibody; or ipilimumab, a fully human human IgG1 monoclonalantibody.

The anti-killer-cell immunoglobulin-like receptors (KIR) antibody may beLirilumab (BMS-986015; developed by Innate Pharma and licenced toBristol-Myers Squibb), a fully human monoclonal antibody.

The anti-LAG-3 antibody is directed against lymphocyte activationgene-3. One such antibody that may be used according to the presentinvention is the monoclonal antibody BMS-986016 (pembrolizumab;Humanized IgG4; Merck).

The anti-4-1BB antibody may be PF-05082566 (Pfizer Oncology), a fullyhumanized IgG2 agonist monoclonal antibody; or Urelumab (BMS-663513;Bristol-Myers Squibb), a fully human IgG4 monoclonal antibody, targeting4-1BB.

In certain embodiments, combinations of antibodies may be used such asbut not limited to: CT-011 in combination with Rituximab (trade namesRituxan, MabThera and Zytux) a chimeric monoclonal antibody against theprotein CD20, for example, each at 3 mg/kg; BMS-936558 (for example 1mg/kg) in combination with ipilimumab; for example at 3 mg/kg); orBMS-936558 (e.g. 1-10 mg/kg) in combination with a anHLA-A*0201-restricted multipeptide vaccine (Weber et al, 2013).

TABLE 1* C646 C375 C146

*Based on Bowers et al. (2010)

In certain embodiments, the agent is a p300 inhibitor, which formulasare listed in Table 1, i.e. C646(4-(4-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoicacid), C146(4-hydroxy-3-(((2-(3-iodophenyl)benzo[d]oxazol-5-yl)imino)methyl)benzoicacid) or C375(2-chloro-4-(5-((2,4-dioxo-3-(2-oxo-2-(p-tolylamino)ethyl)thiazolidin-5-ylidene)methyl)furan-2-yl)benzoicacid). In particular, the p300 inhibitor is C646.

In certain embodiments, the small molecule inhibitor of theindoleamine-2,3-dioxygenase pathway may be Indoximod (NLG-9189; NewLinkGenetics), INCB024360 (Incyte) or NLG-919 (NewLink Genetics).

The HIF-1 regulator may be M30,5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline described inZheng et al. (Zheng et al, 2015).

In certain embodiments, the agent can be derived from a broad spectrumof antibiotics which targets gram-positive and gram-negative bacteria,and thereby facilitating immunomodulation of Tregs, e.g. vancomycinwhich targets gram-positive bacteria and has been shown to reduce Treglevels/activity (Brestoff & Artis, 2013; Smith et al, 2013).

In certain embodiments, the agent may be any copolymer that in a certainregimen will lead to down regulation of Tregs such as YFAK, VYAK, VWAK,VEAK, FEAK, FAK, VAK or WAK. As used herein, the terms “Cop-1” and“Copolymer 1” are used interchangeably.

The pharmaceutical composition of the invention may comprise as activeagent a random copolymer that modulates Treg activity or levelcomprising a suitable quantity of a positively charged amino acid suchas lysine or arginine, in combination with a negatively charged aminoacid (preferably in a lesser quantity) such as glutamic acid or asparticacid, optionally in combination with a non-charged neutral amino acidsuch as alanine or glycine, serving as a filler, and optionally with anamino acid adapted to confer on the copolymer immunogenic properties,such as an aromatic amino acid like tyrosine or tryptophan. Suchcompositions may include any of those copolymers disclosed in WO00/05250, the entire contents of which being hereby incorporated hereinby reference.

More specifically, the composition for use in the present inventioncomprises at least one copolymer selected from the group consisting ofrandom copolymers comprising one amino acid selected from each of atleast three of the following groups: (a) lysine and arginine; (b)glutamic acid and aspartic acid; (c) alanine and glycine; and (d)tyrosine and tryptophan.

The copolymers for use in the present invention can be composed of L- orD-amino acids or mixtures thereof. As is known by those of skill in theart, L-amino acids occur in most natural proteins. However, D-aminoacids are commercially available and can be substituted for some or allof the amino acids used to make the terpolymers and other copolymersused in the present invention. The present invention contemplates theuse of copolymers containing both D- and L-amino acids, as well ascopolymers consisting essentially of either L- or D-amino acids.

In certain embodiments, the pharmaceutical composition of the inventioncomprises Copolymer 1, a mixture of random polypeptides consistingessentially of the amino acids L-glutamic acid (E), L-alanine (A),L-tyrosine (Y) and L-lysine (K) in an approximate ratio of1.5:4.8:1:3.6, having a net overall positive electrical charge and of amolecular weight from about 2 KDa to about 40 KDa. In certainembodiments, the Cop 1 has average molecular weight of about 2 KDa toabout 20 KDa, of about 4.7 KDa to about 13 K Da, of about 4 KDa to about8.6 KDa, of about 5 KDa to 9 KDa, or of about 6.25 KDa to 8.4 KDa. Inother embodiments, the Cop 1 has average molecular weight of about 13KDa to about 20 KDa, of about 13 KDa to about 16 KDa or of about 15 KDato about 16 KDa. Other average molecular weights for Cop 1, lower than40 KDa, are also encompassed by the present invention. Copolymer 1 ofsaid molecular weight ranges can be prepared by methods known in theart, for example by the processes described in U.S. Pat. No. 5,800,808,the entire contents of which are hereby incorporated by reference in theentirety. The Copolymer 1 may be a polypeptide comprising from about 15to about 100, or from about 40 to about 80, amino acids in length. Incertain embodiments, the Cop 1 is in the form of its acetate salt knownunder the generic name glatiramer acetate, that has been approved inseveral countries for the treatment of multiple sclerosis (MS) under thetrade name, Copaxone® (a trademark of Teva Pharmaceuticals Ltd., PetachTikva, Israel). The activity of Copolymer 1 for the pharmaceuticalcomposition disclosed herein is expected to remain if one or more of thefollowing substitutions is made: aspartic acid for glutamic acid,glycine for alanine, arginine for lysine, and tryptophan for tyrosine.

In certain embodiments of the invention, the copolymer that modulatesTreg activity or level is a copolymer of three different amino acidseach from a different one of three groups of the groups (a) to (d).These copolymers are herein referred to as terpolymers.

In one embodiment, the copolymer that modulates Treg activity or levelis a terpolymer containing tyrosine, alanine, and lysine, hereinafterdesignated YAK, in which the average molar fraction of the amino acidscan vary: tyrosine can be present in a mole fraction of about0.05-0.250; alanine in a mole fraction of about 0.3-0.6; and lysine in amole fraction of about 0.1-0.5. The molar ratios of tyrosine, alanineand lysine may be about 0.10:0.54:0.35, respectively. It is possible tosubstitute arginine for lysine, glycine for alanine, and/or tryptophanfor tyrosine.

In certain embodiments, the copolymer that modulates Treg activity orlevel is a terpolymer containing tyrosine, glutamic acid, and lysine,hereinafter designated YEK, in which the average molar fraction of theamino acids can vary: glutamic acid can be present in a mole fraction ofabout 0.005-0.300, tyrosine can be present in a mole fraction of about0.005-0.250, and lysine can be present in a mole fraction of about0.3-0.7. The molar ratios of glutamic acid, tyrosine, and lysine may beabout 0.26:0.16:0.58, respectively. It is possible to substituteaspartic acid for glutamic acid, arginine for lysine, and/or tryptophanfor tyrosine.

In certain embodiments, the copolymer that modulates Treg activity orlevel is a terpolymer containing lysine, glutamic acid, and alanine,hereinafter designated KEA, in which the average molar fraction of theamino acids can vary: glutamic acid can be present in a mole fraction ofabout 0.005-0.300, alanine in a mole fraction of about 0.005-0.600, andlysine can be present in a mole fraction of about 0.2-0.7. The molarratios of glutamic acid, alanine and lysine may be about 0.15:0.48:0.36,respectively. It is possible to substitute aspartic acid for glutamicacid, glycine for alanine, and/or arginine for lysine.

In certain embodiments, the copolymer that modulates Treg activity orlevel is a terpolymer containing tyrosine, glutamic acid, and alanine,hereinafter designated YEA, in which the average molar fraction of theamino acids can vary: tyrosine can be present in a mole fraction ofabout 0.005-0.250, glutamic acid in a mole fraction of about0.005-0.300, and alanine in a mole fraction of about 0.005-0.800. Themolar ratios of glutamic acid, alanine, and tyrosine may be about0.21:0.65:0.14, respectively. It is possible to substitute tryptophanfor tyrosine, aspartic acid for glutamic acid, and/or glycine foralanine.

The average molecular weight of the terpolymers YAK, YEK, KEA and YEAcan vary between about 2 KDa to 40 KDa, preferably between about 3 KDato 35 KDa, more preferably between about 5 KDa to 25 KDa.

Copolymer 1 and the other copolymers that modulates Treg activity orlevel may be prepared by methods known in the art, for example, undercondensation conditions using the desired molar ratio of amino acids insolution, or by solid phase synthetic procedures. Condensationconditions include the proper temperature, pH, and solvent conditionsfor condensing the carboxyl group of one amino acid with the amino groupof another amino acid to form a peptide bond. Condensing agents, forexample dicyclohexylcarbodiimide, can be used to facilitate theformation of the peptide bond. Blocking groups can be used to protectfunctional groups, such as the side chain moieties and some of the aminoor carboxyl groups against undesired side reactions.

For example, the copolymers can be prepared by the process disclosed inU.S. Pat. No. 3,849,550, wherein the N-carboxyanhydrides of tyrosine,alanine, γ-benzyl glutamate and N ε-trifluoroacetyl-lysine arepolymerized at ambient temperatures (20° C.-26° C.) in anhydrous dioxanewith diethylamine as an initiator. The γ-carboxyl group of the glutamicacid can be deblocked by hydrogen bromide in glacial acetic acid. Thetrifluoroacetyl groups are removed from lysine by 1M piperidine. One ofskill in the art readily understands that the process can be adjusted tomake peptides and polypeptides containing the desired amino acids, thatis, three of the four amino acids in Copolymer 1, by selectivelyeliminating the reactions that relate to any one of glutamic acid,alanine, tyrosine, or lysine.

The molecular weight of the copolymers can be adjusted duringpolypeptide synthesis or after the copolymers have been made. To adjustthe molecular weight during polypeptide synthesis, the syntheticconditions or the amounts of amino acids are adjusted so that synthesisstops when the polypeptide reaches the approximate length which isdesired. After synthesis, polypeptides with the desired molecular weightcan be obtained by any available size selection procedure, such aschromatography of the polypeptides on a molecular weight sizing columnor gel, and collection of the molecular weight ranges desired. Thecopolymers can also be partially hydrolyzed to remove high molecularweight species, for example, by acid or enzymatic hydrolysis, and thenpurified to remove the acid or enzymes.

In one embodiment, the copolymers with a desired molecular weight may beprepared by a process, which includes reacting a protected polypeptidewith hydrobromic acid to form a trifluoroacetyl-polypeptide having thedesired molecular weight profile. The reaction is performed for a timeand at a temperature which is predetermined by one or more testreactions. During the test reaction, the time and temperature are variedand the molecular weight range of a given batch of test polypeptides isdetermined. The test conditions which provide the optimal molecularweight range for that batch of polypeptides are used for the batch.Thus, a trifluoroacetyl-polypeptide having the desired molecular weightprofile can be produced by a process, which includes reacting theprotected polypeptide with hydrobromic acid for a time and at atemperature predetermined by test reaction. Thetrifluoroacetyl-polypeptide with the desired molecular weight profile isthen further treated with an aqueous piperidine solution to form a lowtoxicity polypeptide having the desired molecular weight.

In certain embodiments, a test sample of protected polypeptide from agiven batch is reacted with hydrobromic acid for about 10-50 hours at atemperature of about 20-28° C. The best conditions for that batch aredetermined by running several test reactions. For example, in oneembodiment, the protected polypeptide is reacted with hydrobromic acidfor about 17 hours at a temperature of about 26° C.

As binding motifs of Cop 1 to MS-associated HLA-DR molecules are known(Fridkis-Hareli et al, 1999), polypeptides having a defined sequence canreadily be prepared and tested for binding to the peptide binding grooveof the HLA-DR molecules as described in the Fridkis-Hareli et al (1999)publication. Examples of such peptides are those disclosed in WO00/05249 and WO 00/05250, the entire contents of which are herebyincorporated herein by reference, and include the peptides of SEQ IDNOs. 1-32 (Table 2).

Such peptides and other similar peptides would be expected to havesimilar activity as Cop 1. Such peptides, and other similar peptides,are also considered to be within the definition of copolymers thatcross-react with CNS myelin antigens and their use is considered to bepart of the present invention.

TABLE 2 SEQ ID NO. Peptide Sequence  1 AAAYAAAAAAKAAAA  2AEKYAAAAAAKAAAA  3 AKEYAAAAAAKAAAA  4 AKKYAAAAAAKAAAA  5 AEAYAAAAAAKAAAA 6 KEAYAAAAAAKAAAA  7 AEEYAAAAAAKAAAA  8 AAEYAAAAAAKAAAA  9EKAYAAAAAAKAAAA 10 AAKYEAAAAAKAAAA 11 AAKYAEAAAAKAAAA 12 EAAYAAAAAAKAAAA13 EKKYAAAAAAKAAAA 14 EAKYAAAAAAKAAAA 15 AEKYAAAAAAAAAAA 16AKEYAAAAAAAAAAA 17 AKKYEAAAAAAAAAA 18 AKKYAEAAAAAAAAA 19 AEAYKAAAAAAAAAA20 KEAYAAAAAAAAAAA 21 AEEYKAAAAAAAAAA 22 AAEYKAAAAAAAAAA 23EKAYAAAAAAAAAAA 24 AAKYEAAAAAAAAAA 25 AAKYAEAAAAAAAAA 26 EKKYAAAAAAAAAAA27 EAKYAAAAAAAAAAA 28 AEYAKAAAAAAAAAA 29 AEKAYAAAAAAAAAA 30EKYAAAAAAAAAAAA 31 AYKAEAAAAAAAAAA 32 AKYAEAAAAAAAAAA

The definition of a “copolymer that modulates Treg activity or level”according to the invention is meant to encompass other synthetic aminoacid copolymers such as the random four-amino acid copolymers describedby Fridkis-Hareli et al., 2002 and U.S. Pat. No. 8,017,125 (ascandidates for treatment of multiple sclerosis), namely copolymers VFAKcomprising amino acids valine (V), phenylalanine (F), alanine (A) andlysine (K); VYAK comprising amino acids valine (V), tyrosine (Y),alanine (A) and lysine (K); VWAK comprising amino acids valine (V),tryptophan (W), alanine (A) and lysine (K); VEAK comprising amino acidsvaline (V), glutamic acid (E), alanine (A) and lysine (K); FEAKcomprising amino acids phenylalanine (F), glutamic acid (E), alanine (A)and lysine (K); FAK comprising amino acids phenylalanine (F), alanine(A) and lysine (K); VAK comprising amino acids valine (V), alanine (A)and lysine (K); and WAK comprising amino acids tryptophan (W), alanine(A) and lysine (K).

The pharmaceutical composition according to the present invention may befor treating a disease, disorder or condition of the CNS that is aneurodegenerative disease, disorder or condition selected fromAlzheimer's disease, amyotrophic lateral sclerosis, Parkinson's diseaseHuntington's disease, primary progressive multiple sclerosis; secondaryprogressive multiple sclerosis, corticobasal degeneration, Rettsyndrome, a retinal degeneration disorder selected from the groupconsisting of age-related macular degeneration and retinitis pigmentosa;anterior ischemic optic neuropathy; glaucoma; uveitis; depression;trauma-associated stress or post-traumatic stress disorder,frontotemporal dementia, Lewy body dementias, mild cognitiveimpairments, posterior cortical atrophy, primary progressive aphasia orprogressive supranuclear palsy. In certain embodiments, the condition ofthe CNS is aged-related dementia.

In certain embodiments, the condition of the CNS is Alzheimer's disease,amyotrophic lateral sclerosis, Parkinson's disease Huntington's disease.

The pharmaceutical composition according to the present invention mayfurther be for treating an injury of the CNS selected from spinal cordinjury, closed head injury, blunt trauma, penetrating trauma,hemorrhagic stroke, ischemic stroke, cerebral ischemia, optic nerveinjury, myocardial infarction, organophosphate poisoning and injurycaused by tumor excision

As stated above, the inventors have found that the present inventionimproves the cognitive function in mice that emulates Alzheimer'sdisease. Thus, the pharmaceutical composition may be for use inimproving CNS motor and/or cognitive function, for example for use inalleviating age-associated loss of cognitive function, which may occurin individuals free of a diagnosed disease, as well as in peoplesuffering from neurodegenerative disease. Furthermore, thepharmaceutical composition may be for use in alleviating loss ofcognitive function resulting from acute stress or traumatic episode. Thecognitive function mentioned herein above may comprise learning, memoryor both.

The term “CNS function” as used herein refers, inter alia, to receivingand processing sensory information, thinking, learning, memorizing,perceiving, producing and understanding language, controlling motorfunction and auditory and visual responses, maintaining balance andequilibrium, movement coordination, the conduction of sensoryinformation and controlling such autonomic functions as breathing, heartrate, and digestion.

The terms “cognition”, “cognitive function” and “cognitive performance”are used herein interchangeably and are related to any mental process orstate that involves but is not limited to learning, memory, creation ofimagery, thinking, awareness, reasoning, spatial ability, speech andlanguage skills, language acquisition and capacity for judgmentattention. Cognition is formed in multiple areas of the brain such ashippocampus, cortex and other brain structures. However, it is assumedthat long term memories are stored at least in part in the cortex and itis known that sensory information is acquired, consolidated andretrieved by a specific cortical structure, the gustatory cortex, whichresides within the insular cortex.

In humans, cognitive function may be measured by any know method, forexample and without limitation, by the clinical global impression ofchange scale (CIBIC-plus scale); the Mini Mental State Exam (MMSE); theNeuropsychiatric Inventory (NPI); the Clinical Dementia Rating Scale(CDR); the Cambridge Neuropsychological Test Automated Battery (CANTAB)or the Sandoz Clinical Assessment-Geriatric (SCAG). Cognitive functionmay also be measured indirectly using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRI), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

An improvement of one or more of the processes affecting the cognitionin a patient will signify an improvement of the cognitive function insaid patient, thus in certain embodiments improving cognition comprisesimproving learning, plasticity, and/or long term memory. The terms“improving” and “enhancing” may be used interchangeably.

The term “learning” relates to acquiring or gaining new, or modifyingand reinforcing, existing knowledge, behaviors, skills, values, orpreferences.

The term “plasticity” relates to synaptic plasticity, brain plasticityor neuroplasticity associated with the ability of the brain to changewith learning, and to change the already acquired memory. One measurableparameter reflecting plasticity is memory extinction.

The term “memory” relates to the process in which information isencoded, stored, and retrieved. Memory has three distinguishablecategories: sensory memory, short-term memory, and long-term memory.

The term “long term memory” is the ability to keep information for along or unlimited period of time. Long term memory comprises two majordivisions: explicit memory (declarative memory) and implicit memory(non-declarative memory). Long term memory is achieved by memoryconsolidation which is a category of processes that stabilize a memorytrace after its initial acquisition. Consolidation is distinguished intotwo specific processes, synaptic consolidation, which occurs within thefirst few hours after learning, and system consolidation, wherehippocampus-dependent memories become independent of the hippocampusover a period of weeks to years.

In an additional aspect, the present invention is directed to a methodfor treating a disease, disorder, condition or injury of the CentralNervous System (CNS) that does not include the autoimmuneneuroinflammatory disease relapsing-remitting multiple sclerosis (RRMS),said method comprising administering to an individual in need thereof apharmaceutical composition according to the present invention as definedabove, wherein said pharmaceutical composition is administered by adosage regime comprising at least two courses of therapy, each course oftherapy comprising in sequence a treatment session followed by aninterval session.

In certain embodiments, the treatment session comprises administeringthe pharmaceutical composition to the individual and the treatmentsession is maintained at least until the level falls below a reference,the administering is paused during the interval session, and theinterval session is maintained as long as the level is below thereference. The reference may be selected from (a) the level of systemicpresence or activity of regulatory T cells or myeloid-derived suppressorcells measured in the most recent blood sample obtained from saidindividual before said administering; or (b) the level of systemicpresence or activity of regulatory T cells or myeloid-derived suppressorcells characteristic of a population of individuals afflicted with adisease, disorder, condition or injury of the CNS.

Alternatively, the treatment session comprises administering thepharmaceutical composition to the individual and the treatment sessionis maintained at least until the systemic presence or level ofIFNγ-producing leukocytes, or the rate of proliferation of leukocytes inresponse to stimulation rises above a reference, the administering ispaused during the interval session, and the interval session ismaintained as long as said level is above said reference, wherein thereference is selected from (a) the level of systemic presence oractivity of IFNγ-producing leukocytes, or the rate of proliferation ofleukocytes in response to stimulation, measured in the most recent bloodsample obtained from said individual before said administering; or (b)the level of systemic presence or activity of IFNγ-producing leukocytes,or the rate of proliferation of leukocytes in response to stimulation,characteristic of a population of individuals afflicted with a disease,disorder, condition or injury of the CNS.

The embodiments above that describe different features of thepharmaceutical composition of the present invention are relevant alsofor the method of the invention, because the method employs the samepharmaceutical composition.

In yet an additional aspect, the present invention provides apharmaceutical composition for use in treating a disease, disorder,condition or injury of the CNS that does not include the autoimmuneneuroinflammatory disease RRMS, said pharmaceutical compositioncomprising an active agent that causes reduction of the level ofsystemic immunosuppression in an individual selected from: (i) anantibody specific for: (a) CD47; (b) OX40; (c) VEGF-A (bevacizumab); (d)CD25; (e) GITR (GITR triggering mAb (DTA-1)); (f) CCR4; or (g)TIM-3/Galectin9; (h) an anti-killer-cell immunoglobulin-like receptor;(i) an anti-LAG-3; or (j) an anti-4-1BB; (ii) any combination of (a) to(j); (iii) any combination of (a) to (j) in combination with an adjuvantsuch as a TLR9 ligand such as CpG; (iv) a protein selected from: (a)Neem leaf glycoprotein (NLGP); or (b) sCTLA-4; (v) a small moleculeselected from: (a) Sunitinib; (b) Polyoxometalate-1 (POM-1); (c)α,β-methyleneadenosine 5′-diphosphate (APCP); (d) arsenic trioxide(As₂O₃); (e) GX15-070 (Obatoclax); (f) a retinoic acid antagonist suchas Ro 41-5253 or LE-135; (g) an SIRPα (CD47) antagonist, such asCV1-hIgG4 as sole agent or in combination with anti-CD47 antibody; (h) aCCR4 antagonist, such as AF399/420/18025 as sole agent or in combinationwith anti-CCR4 antibody; or (i) an adenosin A2B receptor antagonist,such as PSB603; (j) an antagonist of indoleamine-2,3-dioxygenase; (k) anHIF-1 regulator; (vi) a silencing molecule such as miR-126 antisense andanti-galectin-1 (Gal-1); (vii) OK-432; (viii) a combination of IL-12 andanti-CTLA-4; (ix) an antibiotic agent such as vancomycin; or (x) anycombination of (i) to (ix).

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

The following exemplification of carriers, modes of administration,dosage forms, etc., are listed as known possibilities from which thecarriers, modes of administration, dosage forms, etc., may be selectedfor use with the present invention. Those of ordinary skill in the artwill understand, however, that any given formulation and mode ofadministration selected should first be tested to determine that itachieves the desired results.

Methods of administration include, but are not limited to, parenteral,e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal(e.g., oral, intranasal, buccal, vaginal, rectal, intraocular),intrathecal, topical and intradermal routes. Administration can besystemic or local.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the active agent is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; and a glidant, such as colloidal silicondioxide.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compositions may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen free water, before use.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The determination of the doses of the active ingredient to be used forhuman use is based on commonly used practices in the art, and will befinally determined by physicians in clinical trials. An expectedapproximate equivalent dose for administration to a human can becalculated based on the in vivo experimental evidence disclosed hereinbelow, using known formulas (e.g. Reagan-Show et al. (2007) Dosetranslation from animal to human studies revisited. The FASEB Journal22:659-661). According to this paradigm, the adult human equivalent dose(mg/kg body weight) equals a dose given to a mouse (mg/kg body weight)multiplied with 0.081.

The invention will now be illustrated by the following non-limitingexamples.

Examples Materials and Methods

Animals.

5XFAD transgenic mice (Tg6799) that co-overexpress familial AD mutantforms of human APP (the Swedish mutation, K670N/M671L; the Floridamutation, I716V; and the London mutation, V717I) and PS1 (M146L/L286V)transgenes under transcriptional control of the neuron-specific mouseThy-1 promoter (Oakley et al, 2006), and AD double transgenic B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J mice (Borchelt et al, 1997) were purchasedfrom The Jackson Laboratory. Genotyping was performed by PCR analysis oftail DNA, as previously described (Oakley et al, 2006). Heterozygousmutant cx₃cr1^(GFP/+) mice (Jung et al, 2000)(B6.129P-cx3cr1^(tm1Litt)/J, in which one of the CX₃CR1 chemokinereceptor alleles was replaced with a gene encoding GFP) were used asdonors for BM chimeras. Foxp3.LuciDTR mice (Suffner et al, 2010) werebred with 5XFAD mice to enable conditional depletion of Foxp3⁺ Tregs.Animals were bred and maintained by the Animal Breeding Center of theWeizmann Institute of Science. All experiments detailed herein compliedwith the regulations formulated by the Institutional Animal Care and UseCommittee (IACUC) of the Weizmann Institute of Science.

RNA Purification, cDNA Synthesis, and Quantitative Real-Time PCRAnalysis.

Total RNA of the hippocampal dentate gyrus (DG) was extracted with TRIReagent (Molecular Research Center) and purified from the lysates usingan RNeasy Kit (Qiagen). Total RNA of the choroid plexus was extractedusing an RNA MicroPrep Kit (Zymo Research). mRNA (1 μg) was convertedinto cDNA using a High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems). The expression of specific mRNAs was assayed usingfluorescence-based quantitative real-time PCR (RT-qPCR). RT-qPCRreactions were performed using Fast-SYBR PCR Master Mix (AppliedBiosystems). Quantification reactions were performed in triplicate foreach sample using the standard curve method. Peptidylprolyl isomerase A(ppia) was chosen as a reference (housekeeping) gene. The amplificationcycles were 95° C. for 5 s, 60° C. for 20 s, and 72° C. for 15 s. At theend of the assay, a melting curve was constructed to evaluate thespecificity of the reaction. For ifn-γ and ppia gene analysis, the cDNAwas pre-amplified in 14 PCR cycles with non-random PCR primers, therebyincreasing the sensitivity of the subsequent real-time PCR analysis,according to the manufacturer's protocol (PreAmp Master Mix Kit; AppliedBiosystems). mRNA expression was determined using TaqMan RT-qPCR,according to the manufacturer's instructions (Applied Biosystems). AllRT-qPCR reactions were performed and analyzed using StepOne softwareV2.2.2 (Applied Biosystems). The following TaqMan Assays-on-Demand™probes were used: Mm02342430_g1 (ppia) and Mm01168134_m1 (ifn-γ). Forall other genes examined, the following primers were used:

ppia forward (SEQ ID NO: 33) 5′-AGCATACAGGTCCTGGCATCTTGT-3′ and reverse(SEQ ID NO: 34) 5′-CAAAGACCACATGCTTGCCATCCA-3′; icam1 forward(SEQ ID NO: 35) 5′-AGATCACATTCACGGTGCTGGCTA-3′ and reverse(SEQ ID NO: 36) 5′-AGCTTTGGGATGGTAGCTGGAAGA-3′; vcam1 forward(SEQ ID NO: 37) 5′-TGTGAAGGGATTAACGAGGCTGGA-3′ and reverse(SEQ ID NO: 38) 5′-CCATGTTTCGGGCACATTTCCACA-3′; cxcl10 forward(SEQ ID NO: 39) 5′-AACTGCATCCATATCGATGAC-3′ and reverse (SEQ ID NO: 40)5′-GTGGCAATGATCTCAACAC-3′; ccl2 forward (SEQ ID NO: 41)5′-CATCCACGTGTTGGCTCA-3′ and reverse (SEQ ID NO: 42)5′-GATCATCTTGCTGGTGAATGAGT-3′; tnf-γ forward (SEQ ID NO: 43)5′-GCCTCTTCTCATTCCTGCTT-3′ reverse (SEQ ID NO: 44)CTCCTCCACTTGGTGGTTTG-3′; il-1β forward (SEQ ID NO: 45)5′-CCAAAAGATGAAGGGCTGCTT-3′ and reverse (SEQ ID NO: 46)5′-TGCTGCTGCGAGATTTGAAG-3′; il-12p40 forward (SEQ ID NO: 47)5′-GAAGTTCAACATCAAGAGCA-3′ and reverse (SEQ ID NO: 48)5′-CATAGTCCCTTTGGTCCAG-3′; il-10 forward (SEQ ID NO: 49)5′-TGAATTCCCTGGGTGAGAAGCTGA-3′ and reverse (SEQ ID NO: 50)5′-TGGCCTTGTAGACACCTTGGTCTT-3′; tgfβ2 forward (SEQ ID NO: 51)5′-AATTGCTGCCTTCGCCCTCTTTAC-3′ and reverse (SEQ ID NO: 52)5′-TGTACAGGCTGAGGACTTTGGTGT-3′; igf-1 forward (SEQ ID NO: 53)5′-CCGGACCAGAGACCCTTTG and reverse (SEQ ID NO: 54)5′-CCTGTGGGCTTGTTGAAGTAAAA-3′; bdnf forward (SEQ ID NO: 55)5′-GATGCTCAGCAGTCAAGTGCCTTT-3′ and reverse (SEQ ID NO: 56)5′-GACATGTTTGCGGCATCCAGGTAA-3′;

Immunohistochemistry.

Tissue processing and immunohistochemistry were performed on paraffinembedded sectioned mouse (6 μm thick) and human (10 μm thick) brains.For human ICAM-1 staining, primary mouse anti-ICAM (1:20 Abcam; ab2213)antibody was used. Slides were incubated for 10 min with 3% H2O2, and asecondary biotin-conjugated anti-mouse antibody was used, followed bybiotin/avidin amplification with Vectastain ABC kit (VectorLaboratories). Subsequently, 3,3′-diaminobenzidine (DAB substrate)(Zytomed kit) was applied; slides were dehydrated and mounted withxylene-based mounting solution. For tissue stainings, mice weretranscardially perfused with PBS prior to tissue excision and fixation.CP tissues were isolated under a dissecting microscope (Stemi DV4;Zeiss) from the lateral, third, and fourth ventricles of the brain. Forwhole mount CP staining, tissues were fixated with 2.5% paraformaldehyde(PFA) for 1 hour at 4° C., and subsequently transferred to PBScontaining 0.05% sodium azide. Prior to staining, the dissected tissueswere washed with PBS and blocked (20% horse serum, 0.3% Triton X-100,and PBS) for 1 h at room temperature. Whole mount staining with primaryantibodies (in PBS containing 2% horse serum and 0.3% Triton X-100), orsecondary antibodies, was performed for 1 h at room temperature. Eachstep was followed by three washes in PBS. The tissues were applied toslides, mounted with Immu-mount (9990402, from Thermo Scientific), andsealed with cover-slips. For staining of sectioned brains, two differenttissue preparation protocols (paraffin embedded or microtomedfree-floating sections) were applied, as previously described (Baruch etal, 2013; Kunis et al, 2013). The following primary antibodies wereused: mouse anti-Aβ (1:300, Covance, #SIG-39320); rabbit anti-GFP(1:100, MBL, #598); rat anti-CD68 (1:300, eBioscience, #14-0681); ratanti-ICAM-1 (1:200, Abcam, #AB2213); goat anti-GFP (1:100, Abcam,#ab6658); rabbit anti-IBA-1 (1:300, Wako, #019-19741); goat anti-IL-10(1:20, R&D systems, #AF519); rat anti-Foxp3 (1:20, eBioscience,#13-5773-80); rabbit anti-CD3 (1:500, Dako, #IS503); mouse anti-ZO-1,mouse anti-E-Cahedrin, and rabbit anti-Claudin-1 (all 1:100, Invitrogen,#33-9100, #33-4000, #51-9000); rabbit anti-GFAP (1:200, Dako, #Z0334).Secondary antibodies included: Cy2/Cy3/Cy5-conjugated donkeyanti-mouse/goat/rabbit/rat antibodies (1:200; all from JacksonImmunoresearch). The slides were exposed to Hoechst nuclear staining(1:4000; Invitrogen Probes) for 1 min. Two negative controls wereroutinely used in immunostaining procedures, staining with isotypecontrol antibody followed by secondary antibody, or staining withsecondary antibody alone. For Foxp3 intracellular staining, antigenretrieval from paraffin-embedded slides was performed using RetreivagenKit (#550524, #550527; BD Pharmingen™) Microscopic analysis, wasperformed using a fluorescence microscope (E800; Nikon) orlaser-scanning confocal microscope (Carl Zeiss, Inc.). The fluorescencemicroscope was equipped with a digital camera (DXM 1200F; Nikon), andwith either a 20×NA 0.50 or 40×NA 0.75 objective lens (Plan Fluor;Nikon). The confocal microscope was equipped with LSM 510 laser scanningcapacity (three lasers: Ar 488, HeNe 543, and HeNe 633). Recordings weremade on postfixed tissues using acquisition software (NIS-Elements, F3[Nikon] or LSM [Carl Zeiss, Inc.]). For quantification of stainingintensity, total cell and background staining was measured using ImageJsoftware (NIH), and intensity of specific staining was calculated, aspreviously described (Burgess et al, 2010). Images were cropped, merged,and optimized using Photoshop CS6 13.0 (Adobe), and were arranged usingIllustrator CS5 15.1 (Adobe).

Paraffin Embedded Sections of Human CP.

Human brain sections of young and aged postmortem non-CNS-diseaseindividuals, as well as AD patients, were obtained from the Oxford BrainBank (formerly known as the Thomas Willis Oxford Brain Collection(TWOBC)) with appropriate consent and Ethics Committee approval (TW220).The experiments involving these sections were approved by the WeizmannInstitute of Science Bioethics Committee.

Flow Cytometry, Sample Preparation and Analysis.

Mice were transcardially perfused with PBS, and tissues were treated aspreviously described (Baruch et al, 2013). Brains were dissected and thedifferent brain regions were removed under a dissecting microscope(Stemi DV4; Zeiss) in PBS, and tissues were dissociated using thegentleMACS™ dissociator (Miltenyi Biotec). Choroid plexus tissues wereisolated from the lateral, third and fourth ventricles of the brain,incubated at 37° C. for 45 min in PBS (with Ca²⁺/Mg²⁺) containing 400U/ml collagenase type IV (Worthington Biochemical Corporation), and thenmanually homogenized by pipetting. Spleens were mashed with the plungerof a syringe and treated with ACK (ammonium chloride potassium) lysingbuffer to remove erythrocytes. In all cases, samples were stainedaccording to the manufacturers' protocols. All samples were filteredthrough a 70 μm nylon mesh, and blocked with anti-Fc CD16/32 (1:100; BDBiosciences). For intracellular staining of IFN-γ, the cells wereincubated with para-methoxyamphetamine (10 ng/ml; Sigma-Aldrich) andionomycin (250 ng/ml; Sigma-Aldrich) for 6 h, and Brefeldin-A (10 μg/ml;Sigma-Aldrich) was added for the last 4 h. Intracellular labeling ofcytokines was done with BD Cytofix/Cytoperm™ Plusfixation/permeabilization kit (cat. no. 555028). For Treg staining, aneBioscience FoxP3 staining buffer set (cat. no. 00-5523-00) was used.The following fluorochrome-labeled monoclonal antibodies were purchasedfrom BD Pharmingen, BioLegend, R&D Systems, or eBiosciences, and usedaccording to the manufacturers' protocols: PE or Alexa Fluor450-conjugated anti-CD4; PE-conjugated anti-CD25; PerCP-Cy5.5-conjugatedanti-CD45; FITC-conjugated anti-TCRβ; APC-conjugated anti-IFN-γ;APC-conjugated anti-FoxP3; Brilliant-violet-conjugated anti-CD45. Cellswere analyzed on an LSRII cytometer (BD Biosciences) using FlowJosoftware. In each experiment, relevant negative control groups, positivecontrols, and single stained samples for each tissue were used toidentify the populations of interest and to exclude other populations.

Preparation of BM Chimeras.

BM chimeras were prepared as previously described (Shechter et al, 2009;Shechter et al, 2013). In brief, gender-matched recipient mice weresubjected to lethal whole-body irradiation (950 rad) while shielding thehead (Shechter et al, 2009). The mice were then injected intravenouslywith 5×10⁶ BM cells from CX₃CR1^(GFP/+) donors. Mice were left for 8-10weeks after BM transplantation to enable reconstitution of thehematopoietic lineage, prior to their use in experiments. The percentageof chimerism was determined by FACS analysis of blood samples accordingto percentages of GFP expressing cells out of circulating monocytes(CD11b⁺). In this head-shielded model, an average of 60% chimerism wasachieved, and CNS-infiltrating GFP myeloid cells were verified to beCD45^(high)/CD11b^(high), representing monocyte-derived macrophages andnot microglia (Shechter et al, 2013).

Morris Water Maze.

Mice were given three trials per day, for 4 consecutive days, to learnto find a hidden platform located 1.5 cm below the water surface in apool (1.1 m in diameter). The water temperature was kept between 21-22°C. Water was made opaque with milk powder. Within the testing room, onlydistal visual shape and object cues were available to the mice to aid inlocation of the submerged platform. The escape latency, i.e., the timerequired to find and climb onto the platform, was recorded for up to 60s. Each mouse was allowed to remain on the platform for 15 s and wasthen removed from the maze to its home cage. If the mouse did not findthe platform within 60 s, it was manually placed on the platform andreturned to its home cage after 15 s. The inter-trial interval for eachmouse was 10 min. On day 5, the platform was removed, and mice weregiven a single trial lasting 60 s without available escape. On days 6and 7, the platform was placed in the quadrant opposite the originaltraining quadrant, and the mouse was retrained for three sessions eachday. Data were recorded using the EthoVision V7.1 automated trackingsystem (Noldus Information Technology). Statistical analysis wasperformed using analysis of variance (ANOVA) and the Bonferroni post-hoctest. All MWM testing was performed between 10 a.m. and 5 μm. during thelights-off phase.

Radial Arm Water Maze.

The radial-arm water maze (RAWM) was used to test spatial learning andmemory, as was previously described in detail (Alamed et al, 2006).Briefly, six stainless steel inserts were placed in the tank, formingsix swim arms radiating from an open central area. The escape platformwas located at the end of one arm (the goal arm), 1.5 cm below the watersurface, in a pool 1.1 m in diameter. The water temperature was keptbetween 21-22° C. Water was made opaque with milk powder. Within thetesting room, only distal visual shape and object cues were available tothe mice to aid in location of the submerged platform. The goal armlocation remained constant for a given mouse. On day 1, mice weretrained for 15 trials (spaced over 3 h), with trials alternating betweena visible and hidden platform, and the last 4 trails with hiddenplatform only. On day 2, mice were trained for 15 trials with the hiddenplatform. Entry into an incorrect arm, or failure to select an armwithin 15 sec, was scored as an error. Spatial learning and memory weremeasured by counting the number of arm entry errors or the escapelatency of the mice on each trial. Training data were analyzed as themean errors or escape latency, for training blocks of three consecutivetrials.

GA Administration.

Each mouse was subcutaneously (s.c.) injected with a total dose of 100μg of GA (batch no. P53640; Teva Pharmaceutical Industries, Petah Tiqva,Israel) dissolved in 200 μl of PBS. Mice were either injected accordingto a weekly-GA regimen (Butovsky et al, 2006), or daily-GAadministration (FIG. 8 and FIG. 16). Mice were euthanized either 1 weekafter the last GA injection, or 1 month after treatment, as indicatedfor each experiment.

Conditional Ablation of Treg.

Diphtheria toxin (DTx; 8 ng/g body weight; Sigma) was injectedintraperitoneally (i.p.) daily for 4 consecutive days to Foxp3.LuciDTRmice (Suffner et al, 2010). The efficiency of DTx was confirmed by flowcytometry analysis of immune cells in the blood and spleen, achievingalmost complete (>99%) depletion of the GFP-expressing FoxP3⁺CD4⁺ Tregcells (FIG. 4).

P300 Inhibition.

Inhibition of p300 in mice was performed similarly to previouslydescribed (Liu et al, 2013). p300i (C646; Tocris Bioscience) wasdissolved in DMSO and injected i.p. daily (8.9 mg kg⁻¹ d⁻¹, i.p.) for 1week. Vehicle-treated mice were similarly injected with DMSO.

ATRA Treatment.

All-trans retinoic acid (ATRA) administration to mice was performedsimilarly to previously described (Walsh et al, 2014). ATRA (Sigma) wasdissolved in DMSO and injected i.p. (8 mg kg⁻¹ d⁻¹) every other day overthe course of 1 week. Vehicle-treated mice were similarly injected withDMSO.

Soluble Aβ (sAβ) protein isolation and quantification. Tissuehomogenization and sAβ protein extraction was performed as previouslydescribed (Schmidt et al, 2005). Briefly, cerebral brain parenchyma wasdissected and snap-frozen and kept at −80° C. until homogenization.Proteins were sequentially extracted from samples to obtain separatefractions containing proteins of differing solubility. Samples werehomogenized in 10 volumes of ice-cold tissue homogenization buffer,containing 250 mM of sucrose, 20 mM of Tris base, 1 mM ofethylenediaminetetraacetic acid (EDTA), and 1 mM of ethylene glycoltetraacetic acid (pH 7.4), using a ground glass pestle in a Douncehomogenizer. After six strokes, the homogenate was mixed 1:1 with 0.4%diethylamine (DEA) in a 100-mM NaCl solution before an additional sixstrokes, and then centrifuged at 135,000 g at 4° C. for 45 min. Thesupernatant (DEA-soluble fraction containing extracellular and cytosolicproteins) was collected and neutralized with 10% of 0.5Mof Tris-HCl (pH6.8). Aβ₁₋₄₀ and Aβ₁₋₄₂ were individually measured by enzyme-linkedimmunosorbent assay (ELISA) from the soluble fraction using commerciallyavailable kits (Biolegend; #SIG-38954 and #SIG-38956, respectively)according to the manufacturer instructions.

Aβ Plaque Quantitation.

From each brain, 6 μm coronal slices were collected, and eight sectionsper mouse, from four different pre-determined depths throughout theregion of interest (dentate gyrus or cerebral cortex) wereimmunostained. Histogram-based segmentation of positively stained pixelswas performed using the Image-Pro Plus software (Media Cybernetics,Bethesda, Md., USA). The segmentation algorithm was manually applied toeach image, in the dentate gyrus area or in the cortical layer V, andthe percentage of the area occupied by total Aβ immunostaining wasdetermined. Plaque numbers were quantified from the same 6 μm coronalbrain slices, and are presented as average number of plaques per brainregion. Prior to quantification, slices were coded to mask the identityof the experimental groups, and plaque burden was quantified by anobserver blinded to the identity of the groups.

Statistical Analysis.

The specific tests used to analyze each set of experiments are indicatedin the figure legends. Data were analyzed using a two-tailed Student's ttest to compare between two groups, one-way ANOVA was used to compareseveral groups, followed by the Newman-Keuls post-hoc procedure forpairwise comparison of groups after the null hypothesis was rejected(P<0.05). Data from behavioral tests were analyzed using two-wayrepeated-measures ANOVA, and Bonferroni post-hoc procedure was used forfollow-up pairwise comparison. Sample sizes were chosen with adequatestatistical power based on the literature and past experience, and micewere allocated to experimental groups according to age, gender, andgenotype. Investigators were blinded to the identity of the groupsduring experiments and outcome assessment. All inclusion and exclusioncriteria were pre-established according to the IACUC guidelines. Resultsare presented as means±s.e.m. In the graphs, y-axis error bars represents.e.m. Statistical calculations were performed using the GraphPad Prismsoftware (GraphPad Software, San Diego, Calif.).

Introduction. Alzheimer's disease (AD) is an age-relatedneurodegenerative disease characterized by neuronal damage, amyloid beta(Aβ) plaque formation, and chronic inflammation within the centralnervous system (CNS), leading to gradual loss of cognitive function andbrain tissue destruction (Akiyama et al, 2000; Hardy & Selkoe, 2002).Under these conditions, circulating myeloid cells, and the residentmyeloid cells of the CNS, the microglia, play non-redundant roles inmitigating the neuroinflammatory response (Britschgi & Wyss-Coray, 2007;Cameron & Landreth, 2010; Lai & McLaurin, 2012). Specifically, whereasmicroglia fail to ultimately clear Aβ deposits, CNS-infiltratingmonocyte-derived macrophages (mo-MΦ) play a beneficial role in limitingAβ plaque formation and fighting off AD-like pathology (Butovsky et al,2007; Koronyo-Hamaoui et al, 2009; Mildner et al, 2011; Simard et al,2006; Town et al, 2008). The brain's choroid plexus (CP), whoseepithelial layers form the blood-CSF-barrier (BCSFB), has beenidentified as a selective gateway for leukocyte entry to the CNS,enabling recruitment of mo-MΦ and T cells following neural tissue damage(Kunis et al, 2013; Shechter et al, 2013). Here, we hypothesized that inAD, suboptimal recruitment of inflammation-resolving immune cells to thediseased parenchyma is an outcome of systemic immune failure, involvingCP gateway dysfunction.

Example 1. Choroid Plexus (CP) Gateway Activity Along DiseaseProgression in the Mouse Model of AD

We first examined CP activity along disease progression in the 5XFADtransgenic mouse model of AD (AD-Tg); these mice co-express fivemutations associated with familial AD and develop cerebral Aβ pathologyand gliosis as early as 2 months of age (Oakley et al, 2006). We foundthat along the progressive stages of disease pathology, the CP of AD-Tgmice, compared to age-matched wild-type (WT) controls, expressedsignificantly lower levels of leukocyte homing and traffickingdeterminants, including icam1, vcam1, cxcl10, and ccl2 (FIG. 1A), shownto be upregulated by the CP in response to acute CNS damage, and neededfor transepithelial migration of leukocytes (Kunis et al, 2013; Shechteret al, 2013). Immunohistochemical staining for the integrin ligand,ICAM-1, confirmed its reduced expression by the CP epithelium of AD-Tgmice (FIG. 1b ). In addition, staining for ICAM-1 in human postmortembrains, showed its age-associated reduction in the CP epithelium, inline with our previous observations (Baruch et al, 2014), andquantitative assessment of this effect revealed further decline in ADpatients compared to aged individuals without CNS disease (FIG. 2A).Since the induction of leukocyte trafficking determinants by the CP isdependent on epithelial interferon (IFN)-γ signaling (Kunis et al,2013), we next tested whether the observed effects could reflect loss ofIFN-γ availability at the CP. Examining the CP of 5XFAD AD-Tg mice usingflow cytometry intracellular staining, revealed significantly lowernumbers of IFN-γ-producing cells in this compartment (FIG. 2B), andquantitative real-time PCR (RT-qPCR) analysis confirmed lower mRNAexpression levels of ifn-γ at the CP of AD-Tg mice compared toage-matched WT controls (FIG. 2C).

Example 2. The Functional Relationships Between Treg-Mediated SystemicImmune Suppression, CP Gateway Activity, and AD Pathology

Regulatory T cells (Tregs) play a pivotal role in suppressing systemiceffector immune responses (Sakaguchi et al, 2008). We envisioned thatTreg-mediated systemic immune suppression affects IFN-γ availability atthe CP, and therefore focused on the involvement of Tregs in ADpathology. In line with previous reports of elevated Treg levels andsuppressive activities in AD patients (Rosenkranz et al, 2007; Saresellaet al, 2010; Tones et al, 2013), evaluating Foxp3⁺ Treg frequencies insplenocytes of 5XFAD AD-Tg mice, relative to their age-matched WTlittermates, revealed their elevated levels along disease progression(FIG. 3A, B). To study the functional relationships betweenTreg-mediated systemic immune suppression, CP gateway activity, and ADpathology, we crossbred 5XFAD AD-Tg mice with Foxp3-diphtheria toxinreceptor (DTR⁺) mice, enabling transient conditional in vivo depletionof Foxp3⁺ Tregs in AD-Tg/DTR⁺ mice by administration of diphtheria toxin(DTx) (FIG. 4A). Transient depletion of Tregs resulted in elevated mRNAexpression of leukocyte trafficking molecules by the CP of AD-Tg/DTR⁺mice relative to DTx-treated AD-Tg/DTR⁻ littermates (FIG. 5A). Analysisof the long-term effect of the transient Treg depletion on the brainparenchyma (3 weeks later), revealed immune cell accumulation in thebrain, including elevated numbers of CD45^(high)/CD11b^(high) myeloidcells, representing infiltrating mo-MΦ (Shechter et al, 2013), and CD4⁺T cells (FIG. 5B). In addition, the short and transient depletion ofTregs resulted in a marked enrichment of Foxp3⁺ Tregs among the CD4⁺ Tcells that accumulated within the brain, as assessed by flow cytometry(FIG. 5C, D). RT-qPCR analysis of the hippocampus showed increasedexpression of foxp3 and WO mRNA (FIG. 5E).

We next examined whether the short-term depletion of Tregs, which wasfollowed by accumulation of immunoregulatory cells in sites of brainpathology, led to a long-term effect on brain function. We observedreduction in hippocampal gliosis (FIG. 5F), and reduced mRNA expressionlevels of pro-inflammatory cytokines, such as il-12p40 and tnf-α (FIG.5G). Moreover, cerebral Aβ plaque burden in the hippocampal dentategyrus, and the cerebral cortex (5^(th) layer), two brain regionsexhibiting robust Aβ plaque pathology in 5XFAD AD-Tg mice (Oakley et al,2006), was reduced (FIG. 6A, B). Evaluating the effect on cognitivefunction, using the Morris water maze (MWM) test, revealed a significantimprovement in spatial learning and memory in AD-Tg/DTR⁺ mice followingthe Treg depletion, relative to DTx-treated AD-Tg/DTR⁻ aged matchedmice, reaching performance similar to that of WT mice (FIG. 6C-E). Takentogether, these data demonstrated that transiently breakingTreg-mediated systemic immune suppression in AD-Tg mice resulted inaccumulation of inflammation-resolving cells, including mo-MΦ and Tregs,in the brain, and was followed by resolution of the neuroinflammatoryresponse, clearance of AP, and reversal of cognitive decline.

Example 3. Weekly Administration of Copolymer-1 Reduces Treg-MediatedSystemic Immune Suppression, Improves CP Gateway Activity, and MitigatesAD Pathology

To further substantiate the causal nature of the inverse relationshipbetween systemic immune suppression, CP function and AD pathology, wenext made use of the immunomodulatory compound, Glatiramer acetate (GA;also known as Copolymer-1, or Copaxone®), which in a weeklyadministration regimen was found to have a therapeutic effect in theAPP/PS1 mouse model of AD (Butovsky et al, 2006); this effect wasfunctionally associated with mo-MΦ recruitment to cerebral sites ofdisease pathology (Butovsky et al, 2007). Here, we first examinedwhether the CP in APP/PS1 AD-Tg mice, similarly to our observation in5XFAD AD-Tg mice, is also deficient with respect to IFN-γ expressionlevels. We found that in APP/PS1 AD-Tg mice, IFN-γ levels at the CP werereduced relative to age-matched WT controls (FIG. 7A). These resultsencouraged us to test whether the therapeutic effect of weekly-GA inAPP/PS1 mice (Butovsky et al, 2006), could be reproduced in 5XFAD AD-Tgmice, and if so, whether it would affect systemic Tregs, and activationof the CP for mo-MΦ trafficking. We therefore treated 5XFAD AD-Tg micewith a weekly administration regimen of GA over a period of 4 weeks(henceforth, “weekly-GA”; schematically depicted in FIG. 8A). We foundthat 5XFAD AD-Tg mice treated with weekly-GA, showed reducedneuroinflammation (FIGS. 8B-D), and improved cognitive performance,which lasted up to 2 months after the treatment (FIG. 8E-I). Examiningby flow cytometry the effect of weekly-GA on systemic immunity and onthe CP, we found reduced splenocyte Foxp3⁺ Treg levels (FIG. 9A), and anincrease in IFN-γ-producing cells at the CP of the treated 5XFAD AD-Tgmice, reaching similar levels as those observed in WT controls (FIG.9B). The elevated level of IFN-γ-expressing cells at the CP in theweekly-GA treated mice, was accompanied by upregulated epithelialexpression of leukocyte trafficking molecules (FIG. 9C).

To detect infiltrating mo-MΦ entry to the CNS, we used 5XFADAD-Tg/CX₃CR1^(GFP/+) bone marrow (BM) chimeric mice (prepared using headprotection), allowing the visualization of circulating (greenfluorescent protein (GFP)⁺ labeled) myeloid cells (Shechter et al, 2009;Shechter et al, 2013). We found increased homing of GFP⁺ mo-MΦ to the CPand to the adjacent ventricular spaces following weekly-GA treatment, ascompared to vehicle-treated AD-Tg/CX₃CR1^(GFP/+) controls (FIG. 9D-E).Immunohistochemistry of the brain parenchyma revealed the presence ofGFP⁺ mo-MΦ accumulation at sites of cerebral plaque formation (FIG. 9F),and quantification of infiltrating myeloid cells, by flow cytometryanalysis of the hippocampus in AD-Tg non-chimeric mice, showed increasednumbers of CD11b^(high)CD45^(high)-expressing cells (FIG. 9G, H).Together, these results substantiated the functional linkage betweenmo-MΦ recruitment to sites of AD pathology, reduction of systemic Treglevels and IFN-γ-dependent activation of the CP.

Example 4. A Short-Term Direct Interference with Treg Activity, ImprovesCP Gateway Activity, and Mitigates AD Pathology 4.1 Interference withTreg Activity Using a Small Molecule Histone Acetyltransferase Inhibitor

The findings above, which suggested that Treg-mediated systemic immunesuppression interferes with the ability to fight AD pathology, arereminiscent of the function attributed to Tregs in cancer immunotherapy,in which these cells hinder the ability of the immune system to mount aneffective anti-tumor response (Bos & Rudensky, 2012; Nishikawa &Sakaguchi, 2010). Therefore, we considered that a treatment thatdirectly interferes with Foxp3⁺ Treg cell activity might be advantageousin AD. We tested p300i (C646 (Bowers et al, 2010)), a nonpeptidicinhibitor of p300, a histone acetyltransferase that regulates Tregfunction (Liu et al, 2013); this inhibitor was shown to affect Tregsuppressive activities while leaving protective T effector cellresponses intact (Liu et al, 2013). We found that mice treated withp300i, compared to vehicle (DMSO) treated controls, showed elevatedlevels of systemic IFN-γ-expressing cells in the spleen (FIG. 10A), aswell as in the CP (FIG. 10B). We next treated AD-Tg mice with eitherp300i or vehicle over the course of 1 week, and examined them 3 weekslater for cerebral Aβ plaque burden. Immunohistochemical analysisrevealed a significant reduction in cerebral Aβ plaque load in the p300itreated AD-Tg mice (FIG. 10C-E). We also tested whether the effect onplaque pathology following one course of treatment would last beyond the3 weeks, and if so, whether additional courses of treatment wouldcontribute to a long-lasting effect. We therefore compared AD-Tg micethat received a single course of p300i treatment and were examined 2month later, to an age-matched group that received two courses oftreatments during this period, with a 1-month interval in between(schematically depicted in FIG. 10F). We found that the reduction ofcerebral plaque load was evident even two months after a single courseof treatment, but was stronger in mice that received two courses oftreatments with a 1-month interval in between (FIG. 10G). Since impairedsynaptic plasticity and memory in AD is associated with elevatedcerebral levels of soluble Aβ₁₋₄₀/Aβ₁₋₄₂ (sAβ) levels (Shankar et al,2008), we also measured sAβ levels following a single or repeated cyclesof p300i treatment. Again, we found that both one and two courses (withan interval of 1 month in between) were effective in reducing cerebralsAβ, yet this effect was stronger following repeated courses withrespect to the effect on sAβ₁₄₂ (FIG. 10H). These results indicated thatwhile a single short-term course of treatment is effective, repeatedcourses of treatments would be advantageous to maintain a long-lastingtherapeutic effect, similar to our observations following weekly-GAtreatment.

4.2 Interference with Treg Activity Using an Anti-PD1 Antibody

At 10 months of age, 5XFAD Alzheimer's′ disease (AD) transgenic (Tg)mice were injected i.p. with either 250 ug of anti-PD1 (RMP1-14;#BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;#BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1 and day 4of the experiment, and were examined 3 weeks after (schematicallydepicted in FIG. 11A) for their cognitive performance by radial armwater maze (RAWM) spatial learning and memory task, as was previouslydescribed in detail (Alamed et al, 2006). Briefly, on day 1 of the RAWMtask, mice were trained for 15 trials (spaced over 3 h), with trialsalternating between a visible and hidden platform, and the last 4 trailswith hidden platform only. On day 2, mice were trained for 15 trialswith the hidden platform. Entry into an incorrect arm, or failure toselect an arm within 15 sec, was scored as an error. Spatial learningand memory were measured by counting the number of arm entry errors orthe escape latency of the mice on each trial. Age matched untreated WTand AD-Tg mice were used as controls. We found that 5XFAD AD-Tg micetreated with one treatment session, n which included two injections ofanti-PD1 (on day 1 and day 4), showed, as assessed 3 weeks after,significant improved spatial cognitive performance in the RAWM (FIG.11B).

We next examined whether the effect on disease pathology was associatedwith reduction of systemic immune suppression. We repeated theexperiment described above, this time examining the mice at the end oftreatment session (day 7 of the experiment; schematically depicted inFIG. 12A). We observed that in this time point, attenuation of systemicimmune suppression in PD-1-treated AD-Tg mice, was accompanied by asystemic effect of elevation of IFN-γ-producing CD4 splenocytes (FIG.12B), which correlated to a local effect at the CP of elevation of IFN-γmRNA levels (FIG. 13A), and elevation in expression of CP leukocytetrafficking molecules, the chemokines CCL2 and CXCL10 (FIG. 13B). Thesedata showed that the short session of anti-PD-1 treatment in AD-Tg micewas associated with a systemic response of attenuating Treg-mediatedimmune suppression, as expected (Naidoo et al, 2014), and activation ofthe CP gateway activity for leukocyte trafficking to the CNS.

Finally, we examined the effect on disease pathology in AD-Tg mice, andwhether an additional session of treatment was advantageous in itseffect on pathology. To this end, 10 months old AD-Tg mice, eitherreceived 1 session of anti-PD-1 treatment, as described above, or anadditional treatment with an interval of 3 weeks. Control groups wereeither treated with IgG or untreated, and all groups of mice were testedfor their cognitive performance 3 weeks later (schematically depicted inFIG. 14A). We found that while AD-Tg mice treated with 1 session ofanti-PD-1 (“AD-Tg+PD-1 X1”) and examined 2 months later, displayedsignificant cognitive improvement in comparison to IgG-treated anduntreated AD-Tg mice, the effect was less robust than when the same micewere assessed for the cognitive performance a month earlier. Incontrast, AD-Tg mice which received a second session of anti-PD-1treatment (“AD-Tg+PD-1 X2”) showed significantly better spatial learningand memory abilities in the RAWM compared to AD-Tg which received 1session, as well as compared to IgG-treated or untreated AD-Tg mice(FIG. 14B). These findings revealed that in order to maintain a longlasting therapeutic effect, repeated sessions of are needed.

4.3 Interference with Treg Activity Using a Combination of Anti-PD1Antibody and Anti-CTLA4 Antibody

At 10 months of age, 5XFAD Alzheimer's' disease (AD) transgenic (Tg)mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14; #BE0146;Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4 (InVivoMAbanti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG(IgG2a, #BE0089 or Polyclonal Syrian Hamster IgG, #BE0087; BioxcellLifesciences Pvt. LTD.) antibodies, on day 1 and day 4 of theexperiment, and are examined 3 weeks after for their cognitiveperformance by radial arm water maze (RAWM) spatial learning and memorytask, as described above.

Some mice receive an additional treatment session with an intervalsession of 3 weeks. Control groups are either treated with IgG oruntreated, and all groups of mice are tested for their cognitiveperformance 3 weeks later.

It is expected that the mice treated with the combination of antibodiesdisplay significant cognitive improvement in comparison to IgG-treatedand untreated AD-Tg mice as well as a significant reduction of cerebralplaque load.

Example 5. Augmentation of Treg Activity has an Adverse Effect on ADPathology

To substantiate the negative role of Treg-mediated systemic immunesuppression in AD, we next investigated whether augmenting systemic Treglevels could have an opposite, adverse effect on AD pathology. To testthis, we potentiated Treg-suppressive function in AD-Tg mice byadministration of all-trans retinoic acid (ATRA), which induces Tregdifferentiation (Mucida et al, 2007), stabilizes Treg phenotype (Zhou etal, 2010), and renders Tregs more suppressive (Zhou et al, 2010). Weused 5XFAD AD-Tg mice at relatively early stages of disease progression,and treated them with either ATRA or vehicle (DMSO). ATRA-treated AD-Tgmice showed significantly higher splenocyte frequencies of Foxp3⁺CD25⁺Tregs (FIG. 15A, B). Examining the mice 3 weeks after the last ATRAinjection, revealed a higher cerebral Aβ plaque burden and gliosis(approximately 2-3 fold increase; FIGS. 15C-E), and assessment of sAβrevealed increased cerebral sAβ and sAβ₁₋₄₀/sAβ₁₋₄₂ levels followingaugmentation of systemic Tregs (FIG. 15F-G). Assessment of cognitiveperformance, using the RAWM, showed worsening of the spatial memorydeficits in ATRA-treated AD-Tg mice, relative to vehicle-treated AD-Tgmice (FIG. 15H).

Given our present findings of the negative effect of systemic Tregs onAD pathology, together with the fact that daily administration of GA isknown to induce Tregs and is used in the clinic for treating multiplesclerosis (MS) (Haas et al, 2009; Hong et al, 2005; Weber et al, 2007),we tested whether GA in daily regimen (over a period of 1 month), incontrast to weekly-GA, might have a negative effect on disease pathologyin AD-Tg mice. We compared the effect of daily- vs. weekly-GAadministration (schematically depicted in FIG. 16A), in 5XFAD AD-Tgmice. Assessment of cognitive performance by the RAWM task revealedthat, in contrast to the beneficial effect of weekly-GA treatment,either no beneficial effect on spatial memory, or a tendency towards aworsening effect, was observed in AD-Tg mice that received daily-GA(FIG. 16B). Additionally, unlike the robust effect of the weekly-GAadministration on plaque clearance, daily-GA treated AD-Tg mice did notshow any beneficial effect, or showed a moderate adverse effect onplaque load (FIG. 16C-F). These findings emphasize how MS and AD, twoCNS pathologies associated with neuroinflammation, could be oppositelyand distinctively affected by the same immunomodulatory treatment withdaily-GA (Schwartz & Baruch, 2014a).

Example 6. Direct Interference with Treg Activity Improves CP GatewayActivity and Prevents or Mitigate PTSD Pathology

Severely stressful conditions or chronic stress can lead toposttraumatic stress disorder (PTSD) and depression. We previouslysuggested that CP gateway activity may be critical for coping withmental stress, and in the case of suboptimal function of the CP, mentaltraumatic episode may lead to PTSD (Schwartz & Baruch, 2012). We furtherhypothesized that timely systemic intervention after the trauma, whichwill help modifying the CP response, may prevent development of chronicconditions of PTSD. Our findings that a short term attenuation ofTreg-mediated systemic immune suppression has a long term effect onbrain pathology, suggest that this intervention, immediately followingthe traumatic event, would prevent PTSD development.

To test our working hypothesis that the CP is involved in coping withtraumatic stress and that it might dysfunction in cases where traumaticstress leads to the development of PTSD, we adopted a physiologicalPTSD-like animal model in which the mice exhibit hypervigilantbehaviour, impaired attention, increased risk assessment, and poor sleep(Lebow et al, 2012). In this experimental model of PTSD induction, miceare habituated for 10 days to a reverse day/night cycle, inflicted withtwo episodes of electrical shocks (the trauma and the trigger), referredto as a “PTSD induction”, and evaluated at different time pointssubsequent to trauma. Following the traumatic event mice are injectedwith said compound which transiently reduces peripheral immunesuppression. The mice are treated according to one or more of thefollowing regimens:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks;    -   Mice are injected i.p. with weekly-GA as described above        following the traumatic event, and examined after an interval        session of two weeks;    -   Mice are treated with p300i or vehicle over the course of 1 week        following the traumatic event, and examined 3 weeks later as        described above.        Some mice receive an additional treatment session with an        appropriate interval session.

It is expected that mice that receive the treatment do not displayanxiety behavior associated with PTSD in this experimental model, asassessed by time spent exploring and risk assessing in dark/light mazeor the other behavioral tasks described in (Lebow et al, 2012).

Example 7. Transient Reduction of Systemic Immune Suppression MitigatesParkinson's Disease Pathology

Parkinson disease (PD) transgenic (Tg) mice are used in theseexperiment. The mice are treated at the progressive stages of diseaseaccording to one or more of the following regimens:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks;    -   Mice are injected i.p. with weekly-GA as described above        following the traumatic event, and examined after an interval        session of two weeks;    -   Mice are treated with p300i or vehicle over the course of 1 week        following the traumatic event, and examined 3 weeks later as        described above.        Some mice receive an additional treatment session with an        appropriate interval session (about 3 weeks to one month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that PD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. PD-Tg mice whichreceive two courses of therapy, and examined after an appropriateinterval session are expected to show a long-lasting therapeutic effect.To maintain this therapeutic effect mice are subjected to an activesession of treatment with an appropriate interval session between eachsession.

Example 8. Transient Reduction of Systemic Immune Suppression MitigatesHuntington's Disease Pathology

The model used in these experiments may be the Huntington's disease (HD)R6/2 transgenic mice (Tg) test system. R6/2 transgenic mice over expressthe mutated human huntingtin gene that includes the insertion ofmultiple CAG repeats mice at the progressive stages of disease. Thesemice show progressive behavioral-motor deficits starting as early as 5-6weeks of age, and leading to premature death at 10-13 weeks. Thesymptoms include low body weight, clasping, tremor and convulsions.

The mice are treated according to one or more of the following regimenswhen they are 45 days old:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks.    -   Mice are injected i.p. with weekly-GA as described above        following the traumatic event, and examined after an interval        session of two weeks;    -   Mice are treated with p300i or vehicle over the course of 1 week        following the traumatic event, and examined 3 weeks later as        described above.        Some mice receive an additional treatment session with an        appropriate interval session (about 3 weeks to one month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that HD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. HD-Tg mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession between each session.

Example 9. Transient Reduction of Systemic Immune Suppression MitigatesAmyotrophic Lateral Sclerosis Pathology

The model used in this experiment may be the transgenic miceoverexpressing the defective human mutant SOD1 allele containing theGly93→Ala (G93A) gene (B6SJL-TgN (SOD1-G93A)1Gur (herein “ALS mice”).This model develop motor neuron disease and thus constitute an acceptedanimal model for testing ALS.

The mice are treated according to one or more of the following regimenswhen they are 75 days old:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks.    -   Mice are injected i.p. with weekly-GA as described above        following the traumatic event, and examined after an interval        session of two weeks;    -   Mice are treated with p300i or vehicle over the course of 1 week        following the traumatic event, and examined 3 weeks later as        described above.        Some mice receive an additional treatment session with an        appropriate interval session (about 3 weeks to month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod, or mice are allowed to grasp and hold onto a vertical wire(2 mm in diameter) with a small loop at the lower end. A vertical wireallows mice to use both fore- and hindlimbs to grab onto the wire. Thewire is maintained in a vertically oriented circular motion (the circleradius was 10 cm) at 24 rpm. The time that the mouse is able to hangonto the wire is recorded with a timer.

It is expected that ALS mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. ALS mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession between each session.

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1-43. (canceled)
 44. A method of treating an Alzheimer's Disease, themethod comprising administering to an individual in need thereof acomposition comprising an immune checkpoint inhibitor (or an immunesystem inhibitor) as the only active ingredient, wherein the compositionis administered by a dosage regime comprising at least two courses oftherapy, each course of therapy comprising in sequence a treatmentsession where the composition is administered to the individual followedby a non-treatment period where the composition is not administered tothe individual, wherein the non-treatment period is longer than thetreatment session; wherein, if administration of the composition duringthe treatment session is a repeated administration, the non-treatmentperiod is longer than the period between repeated administrations duringthe treatment session; wherein administration of the compositiontransiently reduces levels of systemic immunosuppression and increaseschoroid plexus gateway activity in facilitating selective recruitment ofimmune cells into the central nervous system, thereby treating theindividual.
 45. The method according to claim 44, wherein theadministration of the composition during the treatment session is asingle administration.
 46. The method according to claim 44, wherein theadministration of the composition during the treatment session is arepeated administration.
 47. The method according to claim 46, whereinthe repeated administration occurs once every two, three, four, five orsix days.
 48. The method according to claim 46, wherein the repeatedadministration occurs once weekly.
 49. The method according to claim 46,wherein the repeated administration occurs once every two, three or fourweeks.
 50. The method according to claim 44, wherein the treatmentsession is from 3 days to four weeks.
 51. The method according to claim50, wherein the treatment session is from one week to four weeks. 52.The method according to claim 44, wherein the non-treatment period isfrom one week to six months.
 53. The method according to claim 52,wherein the non-treatment period is from two weeks to six months. 54.The method according to claim 53, wherein the non-treatment period isfrom three weeks to six months.
 55. The method according to claim 44,wherein the immune checkpoint inhibitor (or the immune system inhibitor)is an antibody or a small molecule that neutralizes or blocks an immunecheckpoint.
 56. The method according to claim 55, wherein the antibodyis a neutralizing or blocking antibody.
 57. The method according toclaim 55, wherein the antibody is an agonistic or activating antibody.58. The method according to claim 55, wherein the antibody is a human orhumanized antibody.
 59. The method according to claim 55, wherein theantibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2antibody, an anti-CTLA-4 antibody, an anti-CD47 antibody, an anti-OX40antibody, an anti-VEGF-A antibody, an anti-CD25 antibody, an anti-GITRantibody, an anti-CCR4 antibody, an anti-TIM-3 antibody, ananti-Galectin9 antibody, an anti-killer-cell immunoglobulin-likereceptors (KIR) antibody, an anti-LAG-3 antibody, an anti-4-1BBantibody, fragment thereof or any combination thereof.
 60. The methodaccording to claim 55, wherein the small molecule is a p300 inhibitor, aSunitinib, a Polyoxometalate-1, α,β-methyleneadenosine 5′-diphosphate,an arsenic trioxide, GX15-070, a retinoic acid antagonist, a SIRPαantagonist, a CCR4 antagonist, an adenosine receptor antagonist, anindoleamine-2,3-dioxygenase antagonist, a HIF-1 regulator or anycombination thereof.
 61. The method according to claim 44, wherein thetransient reduction in the level of systemic immunosuppression isassociated with an increase in a systemic presence or activity ofIFNγ-producing leukocytes and/or an increase in a systemic presence oractivity of an IFNγ cytokine.
 62. The method according to claim 44,wherein the transient reduction in the level of systemicimmunosuppression is associated with an increase in a systemic presenceor activity of effector T cells.
 63. The method according to claim 44,wherein the transient reduction in the level of systemicimmunosuppression is associated with a decrease in a systemic presenceor activity of regulatory T cells and/or a decrease in a systemicpresence of an IL-10 cytokine.
 64. The method according to claim 44,wherein the transient reduction in the level of systemicimmunosuppression is associated with a decrease in a systemic presenceor myeloid-derived suppressor cells (MDSCs).
 65. The method according toclaim 44, wherein the transient reduction in the level of systemicimmunosuppression occurs by release of a restraint imposed on the immunesystem by one or more immune checkpoints.
 66. The method according toclaim 44, wherein the administration of the composition during thetreatment session is maintained at least until a systemic presence oractivity of IFNγ-producing leukocytes and/or an IFNγ cytokine risesabove a reference, at which point the administration is stopped, and thenon-treatment period is maintained as long as the systemic presence oractivity of IFNγ-producing leukocytes and/or an IFNγ cytokine is abovethe reference, wherein the reference includes a) a level of a systemicpresence or activity of IFNγ-producing leukocytes and/or an IFNγcytokine measured in the most recent blood sample obtained from theindividual before the administering; or b) a level of a systemicpresence or activity of IFNγ-producing leukocytes and/or an IFNγcytokine characteristic of a population of individuals afflicted withthe Alzheimer's Disease.
 67. The method according to claim 44, wherein acerebral level of soluble amyloid beta peptide is reduced in theindividual, a cerebral amyloid beta (Aβ) plaque burden is reduced orcleared in the individual, a hippocampal gliosis is reduced in theindividual, a cerebral level of a pro-inflammatory cytokine is reducedin the individual, a brain inflammation is decreased in the individualand/or a cognitive function is improved in the individual.
 68. Themethod according to claim 67, wherein the improved cognitive function islearning, memory, creation of imagery, plasticity, thinking, awareness,reasoning, spatial ability, speech and language skills, languageacquisition, capacity for judgment attention or any combination thereof.69. The method according to claim 44, wherein the immune cells includemonocytes, macrophages, or T cells.
 70. The method according to claim69, wherein the T cells include regulatory T cells.